Methods of identifying inhibitors of FGF23 binding to the binary FGFR-klotho complex for the treatment of hypophosphatemia

ABSTRACT

The present invention is directed to a method of treating hypophosphatemia in a subject. The present invention is also directed to a method of screening for compounds suitable for treatment of hypophosphatemia associated with elevated or normal FGF23. This method involves providing FGF23, FGFR-Klotho complex, and one or more candidate compounds. The FGF23, the FGFR-Klotho complex, and the candidate compounds are combined under conditions effective for the FGF23 and the binary FGFR-Klotho complex to form a ternary complex if present by themselves. This method also involves identifying the candidate compounds, which prevent formation of the complex as being potentially suitable in treating hypophosphatemic conditions associated with elevated or normal FGF23. A method of screening the specificity of compounds which prevent formation of the FGF23-Klotho-FGFR complex is also disclosed.

This application is a division of U.S. patent application Ser. No.12/915,801, filed Oct. 29, 2010, which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/256,361, filed Oct. 30, 2009,each of which is hereby incorporated by reference in its entirety.

The subject matter of this application was made with support from theUnited States Government under National Institutes of Health (NIH) grantnumbers DE13686, AG19712, AG25326, DK48482, DK20543, and DK077276. TheU.S. government has certain rights.

FIELD OF THE INVENTION

The present invention is directed to inhibiting binding of FGF23 to thebinary FGFR-Klotho complex for the treatment of hypophosphatemia.

BACKGROUND OF THE INVENTION

Inorganic phosphate plays a key role in a myriad of biologicalprocesses, including bone mineralization, reversible regulation ofprotein function by phosphorylation, and production of adenosinetriphosphate. Plasma levels of phosphate range between 2.2 and 4.9 mg/dl(Dwyer et al., “Severe Hypophosphatemia in Postoperative Patients,” NutrClin Pract 7(6):279-283 (1992), Alon et al., “Calcimimetics as anAdjuvant Treatment for Familial Hypophosphatemic Rickets,” Clin J Am SocNephrol 3: 658-664 (2008)), and are primarily regulated by modifyingrenal tubular reabsorption. Because of phosphate's pleiotropic activity,imbalances in phosphate homeostasis adversely affect essentially everymajor tissue/organ.

Hypophosphatemia is a common clinical condition with an incidenceranging from 0.2-3.1% in all hospital admissions to 21.5-80% in specificsubgroups of hospitalized patients (Gaasbeek et al., “Hypophosphatemia:An Update on its Etiology and Treatment,” Am J Med 118(10):1094-1101(2005), Brunelli et al., “Hypophosphatemia: Clinical Consequences andManagement,” J Am Soc Nephrol 18(7):1999-2003 (2007)). Acute clinicalmanifestations of hypophosphatemia include respiratory failure, cardiacarrhythmia, hemolysis, rhabdomyolysis, seizures, and coma. Chronicclinical manifestations of hypophosphatemia include myalgia andosteomalacia (Gaasbeek et al., “Hypophosphatemia: An Update on itsEtiology and Treatment,” Am J Med 118(10):1094-1101 (2005)).Hypophosphatemia originates from diverse pathophysiologic mechanisms,most importantly from renal phosphate wasting, an inherited or acquiredcondition in which renal tubular reabsorption of phosphate is impaired(Imel et al., “Fibroblast Growth Factor 23: Roles in Health andDisease,” J Am Soc Nephrol 16(9):2565-2575 (2005); Negri A., “HereditaryHypophosphatemias: New Genes in the Bone-kidney Axis,” Nephrology(Carlton) 12(4):317-320 (2007)). Hypophosphatemia can also be associatedwith alcoholic and diabetic ketoacidosis, acute asthma, chronicobstructive pulmonary disease, sepsis, recovery from organtransplantation, and the “refeeding syndrome”, which refers to metabolicdisturbances seen in malnourished patients on commencing nutrition(Gaasbeek et al., “Hypophosphatemia: An Update on its Etiology andTreatment,” Am J Med 118(10):1094-1101 (2005), Miller et al.,“Hypophosphatemia in the Emergency Department Therapeutics,” Am J EmergMed 18(4):457-461 (2000), Marinella M A., “Refeeding Syndrome andHypophosphatemia,” J Intensive Care Med 20(3):155-159 (2005)).

Oral or intravenous administration of inorganic phosphate salts is thecurrent mainstay for the management of hypophosphatemia. Oral phosphatetherapy requires high doses, which frequently lead to diarrhea orgastric irritation (Shiber et al., “Serum Phosphate Abnormalities in theEmergency Department,” J Emerg Med 23(4):395-400 (2002)). Forintravenous phosphate therapy, the response to any given dose issometimes unpredictable (Bohannon N J., “Large Phosphate Shifts withTreatment for Hyperglycemia,” Arch Intern Med 149(6):1423-1425 (1989),Charron et al., “Intravenous Phosphate in the Intensive Care Unit: MoreAggressive Repletion Regimens for Moderate and Severe Hypophosphatemia,”Intensive Care Med 29(8):1273-1278 (2003); Rosen et al., “IntravenousPhosphate Repletion Regimen for Critically III patients with ModerateHypophosphatemia,” Crit Care Med 23(7):1204-1210 (1995)), andcomplications include “overshoot” hyperphosphatemia, hypocalcemia, andmetastatic calcification (Gaasbeek et al., “Hypophosphatemia: An Updateon its Etiology and Treatment,” Am J Med 118(10):1094-1101 (2005);Shiber et al., “Serum Phosphate Abnormalities in the EmergencyDepartment,” J Emerg Med 23(4):395-400 (2002)). In addition, parenteralregimens are not practical for chronic disorders. Most importantly,replacement therapy alone is never adequate when there is significantrenal phosphate wasting. Therefore, novel strategies for the treatmentof hypophosphatemia are needed.

Kidney transplantation is the preferred treatment of end-stage renalfailure, and hypophosphatemia is a well recognized problem during thefirst weeks after engraftment. The majority of kidney transplantpatients often experience excessive renal phosphate leakage (Schwarz etal., “Impaired Phosphate Handling of Renal Allografts is Aggravatedunder Rapamycin-based Immunosuppression,” Nephrol Dial Transplant 16:378-382 (2001); Moorhead et al., “Hypophosphataemic Osteomalacia afterCadaveric Renal Transplantation,” Lancet 1(7860):694-697 (1974)),because the transplanted kidneys only marginally reabsorb the urinaryphosphate to the circulation. The reasons for this poor reabsorbingactivity on the part of transplanted kidneys are unknown. It frequentlycauses the patients malnutrition and secondary osteoporosis. Thisproblem cannot be treated by a simple exogenous supplementation ofphosphate. Similar renal phosphate leakage with unknown pathology isoften observed in pediatric medicine, with outcomes such as malnutritionor growth retardation.

A recent study in adults demonstrated that as many as 93% of patientsdevelop moderate to severe hypophosphatemia (serum phosphateconcentration 0.9-2.25 mg/dL), an average of 5 weeks followingtransplantation (Ambuhl et al., “Metabolic Aspects of PhosphateReplacement Therapy for Hypophosphatemia After Renal Transplantation:Impact on Muscular Phosphate Content, Mineral Metabolism, and Acid/baseHomeostasis,” Am J Kidney Dis 34:875-83 (1999)).

Health problems associated with circulating phosphate shortage are notlimited to humans. Dairy cows sometimes suffer from hypophosphatemia(too low phosphate in the blood) caused by overproduction of the milk.It not only deteriorates the nutritional quality of the milk but alsooften make the cows useless for milk production. It is a relativelycommon problem in dairy farms (Goff, J P., “Pathophysiology of Calciumand Phosphorus Disorders,” Vet Clin North Am Food Anim Pract16(2):319-37 (2000), Oetzel, G R., “Management of Dry Cows for thePrevention of Milk Fever and Other Mineral Disorders,” Vet Clin North AmFood Anim Pract 16(2):369-86 (2000)).

Fibroblast growth factor (FGF) 23, is an endocrine regulator ofphosphate homeostasis, and was originally identified as the mutated genein patients with the phosphate wasting disorder “autosomal dominanthypophosphatemic rickets” (ADHR) (Anonymous., “Autosomal DominantHypophosphataemic Rickets is Associated with Mutations in FGF23,” NatGenet 26(3):345-348 (2000)). FGF23 inhibits reabsorption of phosphate inthe renal proximal tubule by decreasing the abundance of the type IIsodium-dependent phosphate transporters NaP_(i)-2A and NaP_(i)-2C in theapical brush border membrane (Baum et al., “Effect of Fibroblast GrowthFactor-23 on Phosphate Transport in Proximal Tubules,” Kidney Int68(3):1148-1153 (2005); Perwad et al., “Fibroblast Growth Factor 23Impairs Phosphorus and Vitamin D Metabolism In Vivo and Suppresses25-hydroxyvitamin D-1alpha-hydroxylase Expression In Vitro,” Am JPhysiol Renal Physiol 293(5):F1577-1583 (2007); Larsson et al.,“Transgenic mice expressing fibroblast growth factor 23 under thecontrol of the alpha1(I) collagen promoter exhibit growth retardation,osteomalacia, and disturbed phosphate homeostasis,” Endocrinology145(7):3087-3094 (2004)). The phosphaturic activity of FGF23 isdown-regulated by proteolytic cleavage at the ¹⁷⁶RXXR¹⁷⁹ (SEQ ID NO: 1)motif, where “XX” is defined as “HT”, corresponding to positions 177 and178, respectively, of the FGF23 amino acid sequence, producing aninactive N-terminal fragment (Y25 to R179) and a C-terminal fragment(S180 to I251) (FIG. 1A) (Goetz et al., “Molecular Insights into theKlotho-dependent, Endocrine Mode of Action of Fibroblast Growth Factor19 Subfamily Members,” Mol Cell Biol 27(9):3417-3428 (2007)). FGFreceptor (FGFR) 1 is the principal mediator of the phosphaturic actionof FGF23 (Liu et al., “FGFR3 and FGFR4 do not Mediate Renal Effects ofFGF23,” J Am Soc Nephrol 19(12):2342-2350 (2008); Gattineni et al.,“FGF23 Decreases Renal NaPi-2a and NaPi-2c Expression and InducesHypophosphatemia in vivo Predominantly via FGF Receptor 1,” Am J Physiol297(2):F282-F291 (2009)). In addition, Klotho, a protein first describedas an aging suppressor (Kuro-o et al., “Mutation of the Mouse KlothoGene Leads to a Syndrome Resembling Aging,” Nature 390(6655):45-51(1997)), is required as a coreceptor by FGF23 in its target tissue inorder to exert its phosphaturic activity (Kurosu et al., “Regulation ofFibroblast Growth Factor-23 Signaling by Klotho,” J Biol Chem281(10):6120-6123 (2006); Urakawa et al., “Klotho Converts Canonical FGFReceptor into a Specific Receptor for FGF23,” Nature 444(7120):770-774(2006)). Klotho constitutively binds the cognate FGFRs of FGF23, and thebinary FGFR-Klotho complexes exhibit enhanced binding affinity for FGF23((Kurosu et al., “Regulation of Fibroblast Growth Factor-23 Signaling byKlotho,” J Biol Chem 281(10):6120-6123 (2006); Urakawa et al., “KlothoConverts Canonical FGF Receptor into a Specific Receptor for FGF23,”Nature 444(7120):770-774 (2006)). In co-immunoprecipitation studies, itwas demonstrated that the mature, full-length form of FGF23 (Y25 toI251) but not the inactive N-terminal fragment of proteolytic cleavage(Y25 to R179) binds to binary FGFR-Klotho complexes (Goetz et al.,“Molecular Insights into the Klotho-dependent, Endocrine Mode of Actionof Fibroblast Growth Factor 19 Subfamily Members,” Mol Cell Biol27(9):3417-3428 (2007)).

The present invention is directed to overcoming the deficiencies in theart.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a method of treatinghypophosphatemia in a subject. This method involves selecting a subjectwith hypophosphatemia associated with elevated or normal FGF23 levels,and administering to the selected subject an inhibitor ofFGF23-Klotho-FGF receptor complex formation under conditions effectiveto treat the hypophosphatemia.

A second aspect of the present invention relates to a method ofscreening for compounds suitable for treatment of hypophosphatemiaassociated with elevated or normal FGF23 levels. This method involvesproviding: FGF23, binary FGFR-Klotho complex, and one or more candidatecompounds. The FGF23, the FGFR-Klotho complex, and the candidatecompounds are combined under conditions effective for the FGF23 and thebinary FGFR-Klotho complex to form a ternary complex if present bythemselves. The candidate compounds, which prevent formation of thecomplex, are identified as being potentially suitable in treatinghypophosphatemia associated with elevated or normal FGF23 levels.

The present invention also relates to a method of screening thespecificity of compounds which prevent formation of theFGF23-Klotho-FGFR complex. This method involves providing FGF19,providing binary FGFR-βKlotho complex, and providing one or morecandidate compounds. The FGF19, the binary FGFR-βKlotho complex, and thecandidate compounds are combined under conditions effective for theFGF19 and the binary FGFR-βKlotho complex to form a ternary complex ifpresent by themselves. Candidate compounds which do not interfere withformation of the complex are identified as being specific andpotentially suitable in treating hypophosphatemia associated withelevated or normal FGF23 levels.

Fibroblast growth factor (FGF) 23 is a key hormone and regulator ofphosphate homeostasis, which inhibits renal phosphate reabsorption byactivating FGF receptor (FGFR) 1c in a Klotho-dependent fashion. Thepresent invention shows that proteolytic cleavage at the RXXR motifdown-regulates FGF23's activity by a dual mechanism: by removing thebinding site for the binary FGFR-Klotho complex that resides in theC-terminal region of FGF23, and by generating an endogenous FGF23inhibitor. The soluble ectodomains of FGFR1c and Klotho are sufficientto form a ternary complex with FGF23 in vitro. The C-terminal tail ofFGF23 mediates binding of FGF23 to a de novo site generated at thecomposite FGFR1c-Klotho interface. Consistent with this finding, theisolated 72-residue-long C-terminal tail of FGF23—the C-terminalfragment of proteolytic cleavage at the RXXR motif—impairs FGF23signaling by competing with full-length ligand for binding to the binaryFGFR-Klotho complex. Injection of the FGF23 C-terminal tail peptide intohealthy rats inhibits renal phosphate excretion and induceshyperphosphatemia. In a mouse model of renal phosphate wastingattributable to high FGF23, the FGF23 C-terminal tail peptide reducesphosphate excretion leading to an increase in serum phosphateconcentration. It is proposed that the proteolytic C-terminal fragmentof FGF23 is an endogenous inhibitor of FGF23 and that peptides derivedfrom the C-terminal tail of FGF23, or peptidomimetics and small moleculeorganomimetics of the C-terminal tail can be used as novel therapeuticsto treat hypophosphatemia where FGF23 is not down-regulated as acompensatory mechanism.

Applicants have determined that the 72-amino acid C-terminal tail ofFGF23 mediates binding of FGF23 to the binary FGFR-Klotho complex and,indeed, this region harbors the FGF23-binding site for the binaryFGFR-Klotho complex. Based on this finding, the ability of theC-terminal region of FGF23 to antagonize FGF23 binding to FGFR-Klothoand its phosphaturic action is evaluated. It is shown that peptidesderived from this region are able to competitively displace full-lengthFGF23 from its ternary complex with Klotho and FGFR, and inhibit FGF23signaling. It is further shown that these peptides are able toantagonize FGF23's phosphaturic activity in vivo, both in healthy ratsand in a mouse model of phosphate wasting disorders. Based on thesedata, it is believed that peptides derived from the C-terminal tail ofFGF23, or peptidomimetics and small molecule organomimetics of theC-terminal tail can be used as novel therapeutics to treat patients withhypophosphatemia where FGF23 is not down-regulated as a compensatorymechanism.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-G show that FGF23 binds to the preformed binary complex of theectodomains of FGFR and Klotho. FIG. 1A shows the FGF23 proteins andpeptides used in this study. Amino acid boundaries of eachprotein/peptide are labeled with residue letter and number. The FGF23core region is shaded grey, and the position of the proteolytic cleavagesite RXXR (SEQ ID NO: 1) is indicated, where “XX” is defined as “HT”,corresponding to positions 177 and 178 of SEQ ID NO: 3, respectively, ofthe FGF23 amino acid sequence. FIG. 1B shows a size-exclusionchromatogram of the 1:1 FGFR1c-Klotho complex. Arrows indicate theretention times of molecular size standards and the void volume (V_(V)).Proteins of column peak fractions were resolved on 14%SDS-polyacrylamide gels and stained with Coomassie Blue. FIG. 1C shows asize-exclusion chromatogram of the ternary FGF23²⁸⁻²⁵¹-FGFR1c-Klothocomplex. Arrows indicate the retention times of molecular size standardsand the void volume (V_(V)). Proteins of column peak fractions wereresolved on 14% SDS-polyacrylamide gels and stained with Coomassie Blue.FIG. 1D shows a representative surface plasmon resonance (SPR)sensorgram of FGFR1c binding to Klotho, and fitted saturation bindingcurve. Klotho ectodomain was immobilized on a biosensor chip, andincreasing concentrations of FGFR1c ectodomain were passed over thechip. The dissociation constant (K_(D)) was calculated from thesaturation binding curve. FIG. 1E shows a representative SPR sensorgramillustrating binding of FGF23²⁸⁻²⁵¹ to the binary FGFR1c-Klotho complex.FGF23²⁸⁻²⁵¹ was immobilized on a biosensor chip, and increasingconcentrations of FGFR1c-Klotho complex were passed over the chip. FIG.1F shows a representative SPR sensorgram of FGF23 binding to Klothoalone. FGF23²⁸⁻²⁵¹ was immobilized on a biosensor chip, and increasingconcentrations of Klotho ectodomain were passed over the chip. FIG. 1Gshows a representative SPR sensorgram of FGF23 binding to FGFR1c alone.FGF23²⁸⁻²⁵¹ was immobilized on a biosensor chip, and increasingconcentrations of FGFR1c ectodomain were passed over the chip.

FIGS. 2A-D show the FGF23 C-terminal tail mediates binding of FGF23 tothe binary FGFR-Klotho complex. FIG. 2A shows a representative SPRsensorgram illustrating binding of FGF23¹⁸⁰⁻²⁵¹ to the binaryFGFR1c-Klotho complex. FGF23¹⁸⁰⁻²⁵¹ was immobilized on a biosensor chip,and increasing concentrations of FGFR1c-Klotho complex were passed overthe chip. FIG. 2B shows a size-exclusion chromatogram of the mixture ofthe 1:1 FGFR1c-Klotho complex with FGF23¹⁸⁰⁻²⁵¹. Arrows indicate theretention times of molecular size standards and the void volume (V_(V)).Proteins of column peak fractions were resolved on 14%SDS-polyacrylamide gels and stained with Coomassie Blue. FIG. 2C shows asize-exclusion chromatogram of the mixture of the 1:1 FGFR1c-Klothocomplex with FGF23²⁸⁻¹⁷⁹. Arrows indicate the retention times ofmolecular size standards and the void volume (V_(V)). Proteins of columnpeak fractions were resolved on 14% SDS-polyacrylamide gels and stainedwith Coomassie Blue. FIG. 2D shows analysis of FGF23 protein/peptidebinding to FGFR-Klotho complex by pull-down assay. Lysate of HEK293cells stably expressing Klotho was incubated with FGF23 proteins, orprotein sample buffer (control). Binary complexes of endogenous FGFR andKlotho were isolated from cell lysate by immunoprecipitation (IP) andanalyzed for bound FGF23 protein/peptide.

FIGS. 3A-H show that the isolated FGF23 C-terminal tail peptide competeswith FGF23 for binding to the binary FGFR-Klotho complex. FIG. 3A showsa representative SPR sensorgram illustrating inhibition by FGF23¹⁸⁰⁻²⁵¹of FGFR1c-Klotho binding to FGF23²⁸⁻²⁵¹ immobilized on a biosensor chip.Increasing concentrations of FGF23¹⁸⁰⁻²⁵¹ were mixed with a fixedconcentration of FGFR1c-Klotho complex and the mixtures were passed overa FGF23 chip. FIG. 3B shows a representative SPR sensorgram illustratinginhibition by FGF23¹⁸⁰⁻²⁰⁵ of FGFR1c-Klotho binding to FGF23²⁸⁻²⁵¹immobilized on a biosensor chip. Increasing concentrations ofFGF23¹⁸⁰⁻²⁰⁵ were mixed with a fixed concentration of FGFR1c-Klothocomplex and the mixtures were passed over a FGF23 chip. The sequences ofFGF23¹⁸⁰⁻²⁵¹, FGF23¹⁸⁰⁻²⁰⁵, and FGF23²⁸⁻²⁵¹ are listed in Table 1. FIG.3C shows a representative SPR sensorgram illustrating inhibition byFGF23²⁸⁻²⁵¹ of FGFR1c-Klotho binding to FGF23²⁸⁻²⁵¹ immobilized on abiosensor chip. Increasing concentrations of FGF23²⁸⁻²⁵¹ were mixed witha fixed concentration of FGFR1c-Klotho complex and the mixtures werepassed over a FGF23 chip. FIG. 3D shows dose-response curves forinhibition by FGF23¹⁸⁰⁻²⁵¹ (filled circles), FGF23¹⁸⁰⁻²⁰⁵ (opencircles), or FGF23²⁸⁻²⁵¹ (filled triangles) of FGFR1c-Klotho binding toFGF23 immobilized on a biosensor chip (see also SPR sensorgrams shown inFIGS. 3A-C). For each dose-response curve, averaged data from two tothree SPR experiments are presented. Inhibition of binding by the FGF23C-terminal peptides and full-length FGF23, respectively, is expressed aspercent of the binding response obtained for the binary FGFR1c-Klothocomplex alone, and plotted as a function of the concentration of FGF23protein/peptide. Note that the dose-response curves of the C-terminalFGF23 peptides are shifted to the right by about 3-fold and 6-fold,respectively, compared to the dose-response curve of full-length FGF23.Error bars denote SD. FIG. 3E shows a representative SPR sensorgramillustrating inhibition by FGF23²⁸⁻²⁵¹ of FGFR1c-Klotho binding toFGF23¹⁸⁰⁻²⁵¹ immobilized on a biosensor chip. Increasing concentrationsof FGF23²⁸⁻²⁵¹ were mixed with a fixed concentration of FGFR1c-Klothocomplex and the mixtures were passed over a FGF23¹⁸⁰⁻²⁵¹ chip. FIG. 3Fshows a representative SPR sensorgram illustrating inhibition byFGF23¹⁸⁰⁻²⁵¹ of FGFR1c-Klotho binding to FGF23¹⁸⁰⁻²⁵¹ immobilized on abiosensor chip. Increasing concentrations of FGF23¹⁸⁰⁻²⁵¹ were mixedwith a fixed concentration of FGFR1c-Klotho complex and the mixtureswere passed over a FGF23¹⁸⁰⁻²⁵¹ chip. FIG. 3G shows a representative SPRsensorgram illustrating no inhibition by FGF21¹⁶⁸⁻²⁰⁹ of FGFR1c-Klothobinding to FGF23²⁸⁻²⁵¹ immobilized on a biosensor chip. FGF21¹⁶⁸⁻²⁰⁹ andFGFR1c-Klotho complex were mixed at molar ratios of 6:1 and 10:1, andthe mixtures were passed over a FGF23 chip. FIG. 3H shows inhibition byFGF23¹⁸⁰⁻²⁵¹ of FGFR-Klotho binding to FGF23²⁸⁻²⁵¹ using aco-immunoprecipitation based competition assay. Cognate FGFRs of FGF23were co-immunoprecipitated with Klotho from lysates of a HEK293 cellline stably expressing Klotho (IP) Immunoprecipitated binary FGFR-Klothocomplexes were incubated with either FGF23¹⁸⁰⁻²⁵¹ or FGF23²⁸⁻²⁵¹ alone,or with mixtures of FGF23²⁸⁻²⁵¹ with increasing FGF23¹⁸⁰⁻²⁵¹, andsubsequently analyzed for bound FGF23 protein(s). A 76-fold molar excessof FGF23¹⁸⁰⁻²⁵¹ completely blocked binding of FGF23²⁸⁻²⁵¹ to theFGFR-Klotho complex. Consistent with the data shown in FIGS. 2A-D,FGF23¹⁸⁰⁻²⁵¹ alone co-precipitated with each of the three binaryFGFR-Klotho complexes (first lane of each immunoblot). The sequences ofFGF23¹⁸⁰⁻²⁵¹ FGF23¹⁸⁰⁻²⁰⁵, and FGF23²⁸⁻²⁵¹ are listed in Table 1.

FIGS. 4A-D show that the FGF23 C-terminal tail peptide does notinterfere with binary complex formation between βKlotho and FGF19/FGF21,nor does it interfere with ternary complex formation between βKlotho,FGFR, and FGF19/FGF21. FIG. 4A shows a representative SPR sensorgramillustrating no inhibition by FGF23¹⁸⁰⁻²⁵¹ of βKlotho binding toFGF19²³⁻²¹⁶ immobilized on a biosensor chip. FGF23¹⁸⁰⁻²⁵¹ and βKlothowere mixed at a molar ratio of 2:1, and the mixture was passed over aFGF19 chip. FIG. 4B shows a representative SPR sensorgram illustratingno inhibition by FGF23¹⁸⁰⁻²⁵¹ of βKlotho binding to FGF21²⁹⁻²⁰⁹immobilized on a biosensor chip. FGF23¹⁸⁰⁻²⁵¹ and βKlotho were mixed ata molar ratio of 2:1, and the mixture was passed over a FGF21 chip. FIG.4C shows a representative SPR sensorgram illustrating no inhibition byFGF23¹⁸⁰⁻²⁵¹ of FGFR1c-βKlotho binding to FGF19²³⁻²¹⁶ immobilized on abiosensor chip. FGF23¹⁸⁰⁻²⁵¹ and FGFR1c-βKlotho complex were mixed at amolar ratio of 10:1, and the mixture was passed over a FGF19 chip. FIG.4D shows a representative SPR sensorgram illustrating no inhibition byFGF23¹⁸⁰⁻²⁵¹ of FGFR1c-βKlotho binding to FGF21²⁹⁻²⁰⁹ immobilized on abiosensor chip. FGF23¹⁸⁰⁻²⁵¹ and FGFR1c-βKlotho complex were mixed at amolar ratio of 10:1, and the mixture was passed over a FGF21 chip.

FIGS. 5A-C show that residues S180 to T200 of the C-terminal tail ofFGF23 comprise the minimal binding epitope for the FGFR-Klotho complex.FIG. 5A shows that FGF23²⁸⁻²⁰⁰ induces tyrosine phosphorylation of FRS2αand downstream activation of MAP kinase cascade. Shown is an immunoblotanalysis for phosphorylation of FRS2α (pFRS2α) and 44/42 MAP kinase(p44/42 MAPK) in a CHO Klotho cell line, which had been stimulated witheither FGF23²⁸⁻²⁵¹ or FGF23²⁸⁻²⁰⁰. Numbers above the lanes give theamounts of protein added in nM. To control for equal sample loading, theprotein blots were probed with antibodies to non-phosphorylated^(44/42)MAP kinase (44/42 MAPK) and Klotho. FIG. 5B shows that FGF23²⁸⁻²⁰⁰exhibits phosphaturic activity. FGF23²⁸⁻²⁵¹ and FGF23²⁸⁻²⁰⁰ wereinjected IP into C57BL/6 mice, and serum levels of phosphate (serumP_(i)) were measured before and after FGF23 protein injection. Bars anderror bars denote mean±SE. An asterisk indicates P<0.05 by ANOVA. FIG.5C shows that FGF23¹⁸⁰⁻²⁰⁵—the minimal binding epitope for theFGFR-Klotho complex—competes with FGF23 for binding to FGFR-Klotho.Cognate FGFRs of FGF23 were co-immunoprecipitated with Klotho fromlysates of a HEK293 cell line stably expressing Klotho (IP).Immunoprecipitated binary FGFR-Klotho complexes were incubated witheither FGF23²⁸⁻²⁵¹ alone or mixtures of FGF23²⁸⁻²⁵¹ with increasingFGF23¹⁸⁰⁻²⁰⁵, and subsequently analyzed for bound FGF23 protein(s). TheFGF23¹⁸⁰⁻²⁰⁵ peptide inhibited co-precipitation of FGF23²⁸⁻²⁵¹ with eachof the three binary FGFR-Klotho complexes in a dose-dependent fashion,albeit with over 100-fold reduced potency compared to the FGF23¹⁸⁰⁻²⁵¹peptide (FIG. 3H). The sequences of FGF23¹⁸⁰⁻²⁵¹, FGF23¹⁸⁰⁻²⁰⁵, andFGF23²⁸⁻²⁵¹ are listed in Table 1.

FIGS. 6A-C show that FGF23 C-terminal peptides impair ternary complexformation between FGF23, Klotho, and FGFR, and specifically block FGF23signaling. FIG. 6A shows that FGF23¹⁸⁰⁻²⁵¹ inhibits tyrosinephosphorylation of FRS2α and downstream activation of MAP kinase cascadeinduced by FGF23²⁸⁻²⁵¹. Shown is an immunoblot analysis forphosphorylation of FRS2α (pFRS2α) and 44/42 MAP kinase (p44/42 MAPK) ina HEK293 Klotho cell line, which had been stimulated with FGFproteins/peptide as denoted in the figure. Numbers above the lanes givethe amounts of protein/peptide added in nM. To control for equal sampleloading, the protein blots were probed with an antibody to Klotho. FIG.6B shows that FGF23¹⁸⁰⁻²⁰⁵ inhibits tyrosine phosphorylation of FRS2αand downstream activation of MAP kinase cascade induced by FGF23²⁸⁻²⁵¹.Shown is an immunoblot analysis for phosphorylation of FRS2α (pFRS2α)and 44/42 MAP kinase (p44/42 MAPK) in a HEK293 Klotho cell line, whichhad been stimulated with either FGF23¹⁸⁰⁻²⁰⁵ alone or mixtures ofFGF23²⁸⁻²⁵¹ with increasing FGF23¹⁸⁰⁻²⁰⁵. Numbers above the lanes givethe amounts of peptide added in μM. To control for equal sample loading,the protein blots were probed with an antibody to non-phosphorylated44/42 MAP kinase (44/42 MAPK). FIG. 6C shows that FGF23¹⁸⁰⁻²⁵¹ fails toinhibit tyrosine phosphorylation of FRS2α and downstream activation ofMAP kinase cascade induced by FGF2. Shown is an immunoblot analysis forphosphorylation of FRS2α (pFRS2α) and 44/42 MAP kinase (p44/42 MAPK) ina HEK293 Klotho cell line, which had been stimulated with either FGF2alone or mixtures of FGF2 with increasing FGF23¹⁸⁰⁻²⁵¹. Numbers abovethe lanes give the amounts of peptide added in nM. To control for equalsample loading, the protein blots were probed with an antibody toKlotho. The sequences of FGF23¹⁸⁰⁻²⁵¹, FGF23¹⁸⁰⁻²⁰⁵, and FGF23²⁸⁻²⁵¹ arelisted in Table 1.

FIGS. 7A-B show that FGF23 C-terminal peptides antagonize the inhibitoryeffect of FGF23 on sodium-coupled phosphate uptake. Opossum kidney OKPcells were stimulated with either FGF23²⁸⁻²⁵¹ or FGF23¹⁸⁰⁻²⁵¹ orFGF23¹⁸⁰⁻²⁰⁵ alone, or mixtures of FGF23²⁸⁻²⁵¹ with either increasingFGF23¹⁸⁰⁻²⁵¹ (FIG. 7A) or increasing FGF23¹⁸⁰⁻²⁰⁵ (FIG. 7B). After 4 hcell stimulation, sodium-dependent phosphate uptake was measured. Barsand error bars denote mean+SE. An asterisk indicates P<0.05 by ANOVA.

FIG. 8 shows that the FGF23 C-terminal tail peptide antagonizesphosphaturic activity of FGF23 in vivo. FGF23²⁸⁻²⁵¹ (0.1 μg kg bodyweight⁻¹) or FGF23¹⁸⁰⁻²⁵¹ (0.1 μg kg body weight⁻¹) were injected IVinto Sprague-Dawley rats. Serum and urine parameters were measured andcalculated before and 3 h after injection. FE P_(i): fractionalexcretion of phosphate; U_(Pi)V: phosphate excretion rate; Cl_(Cr):creatinine clearance.

FIGS. 9A-C show that the FGF23 C-terminal tail peptide inhibits theability of FGF23 to down-regulate the expression of the type IIsodium-coupled phosphate transporters NaP_(i)-2A and NaP_(i)-2C in theapical brush border membrane. Sprague-Dawley rats were given IVFGF23²⁸⁻²⁵¹(0.1 μg kg body weight⁻¹), FGF23¹⁸⁰⁻²⁵¹ (0.1 μg kg bodyweight⁻¹), or vehicle, and renal tissue was isolated 3 h post injection.FIG. 9A shows representative images of cryosections of renal tissueprocessed for NaP_(i)-2A immunostaining and β-actin staining. FIGS. 9B-Cshow NaP_(i)-2A (FIG. 9B) and NaP_(i)-2C (FIG. 9C) protein abundance inrenal cortex tissue (cortex) and isolated brush border membrane vesicles(BBMV). Equal amounts of protein were separated by SDS-PAGE and probedfor either NaP_(i)-2A or NaP_(i)-2C, and β-actin by immunoblot.Representative protein blots with tissues from 6 rats are shown in theupper panels of each figure part. Summarized data of renal tissuesamples from 12 rats are presented in the bottom panels. Bars and errorbars are mean+SE. An asterisk denotes P<0.05 by ANOVA.

FIG. 10 shows that FGF23 C-terminal peptides alleviate renal phosphatewasting in Hyp mice. FGF23¹⁸⁰⁻²⁵¹ (1 mg), FGF23¹⁸⁰⁻²⁰⁵ (860 μg), orvehicle were injected IP into Hyp mice. Urine phosphate (urinary P_(i))and creatinine levels and serum phosphate levels (serum P_(i)) weremeasured before and at the indicated time points after the injection.Urinary P_(i) of Hyp mice treated with FGF23¹⁸⁰⁻²⁰⁵ was not determined(ND). Bars and error bars are mean+SE. An asterisk denotes P<0.05 byANOVA, two asterisks denote P<0.01.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to a method of treatinghypophosphatemia in a subject. This method involves selecting a subjectwith hypophosphatemia associated with elevated or normal FGF23 levelsand administering to the selected subject an inhibitor ofFGF23-Klotho-FGF receptor complex formation under conditions effectiveto treat the hypophosphatemia.

As described by Goetz et al. (Goetz et al., “Molecular Insights into theKlotho-Dependent, Endocrine Mode of Action of Fibroblast Growth Factor19 Subfamily Members,” Mol Cell Biol 3417-3428 (2007), which is herebyincorporated by reference in its entirety), the mammalian fibroblastgrowth factor (FGF) family comprises 18 polypeptides (FGF1 to FGF10 andFGF16 to FGF23), which participate in a myriad of biological processesduring embryo genesis, including but not limited to gastrulation, bodyplan formation, somitogenesis, and morphogenesis of essentially everytissue/organ such as limb, lung, brain, and kidney (Bottcher et al.,“Fibroblast Growth Factor Signaling During Early VertebrateDevelopment,” Endocr Rev 26:63-77 (2005), and Thisse et al., “Functionsand Regulations of Fibroblast Growth Factor Signaling During EmbryonicDevelopment,” Dev Biol 287:390-402 (2005), which are hereby incorporatedby reference in their entirety).

FGFs execute their biological actions by binding to, dimerizing, andactivating FGF receptor (FGFR) tyrosine kinases, which are encoded byfour distinct genes (Fgfr1 to Fgfr4). Prototypical FGFRs consist of anextracellular domain composed of three immunoglobulin-like domains, asingle-pass transmembrane domain, and an intracellular domainresponsible for the tyrosine kinase activity (Mohammadi et al.,“Structural Basis for Fibroblast Growth Factor Receptor Activation,”Cytokine Growth Factor Rev 16:107-137 (2005), which is herebyincorporated by reference in its entirety).

FGF23 is a gene cloned by Itoh et al. at Kyoto University (WO 01/66596to Itoh et al., which is hereby incorporated by reference in itsentirety). FGF23 mRNA is expressed mainly in the brain, preferentiallyin the ventrolateral thalamic nucleus. It is also expressed in thethymus at low levels (Yamashita et al., “Identification of a NovelFibroblast Growth Factor, FGF-23, Preferentially Expressed in theVentrolateral Thalamic Nucleus of the Brain,” Biochem Biophys Res Comm277(2):494-498 (2000), which is hereby incorporated by reference in itsentirety). The tissue with the highest level of FGF23 expression is bone(osteocytes and osteoblasts), where it is highly expressed during phasesof active bone remodeling (Riminucci et al., “FGF-23 in FibrousDysplasia of Bone and its Relationship to Renal Phosphate Wasting,” JClin Invest 112:683-692 (2003), which is hereby incorporated byreference in its entirety). Expression of FGF23 in dendritic cells hasalso been reported (Katoh et al., “Comparative Genomics on MammalianFgf6-Fgf23 Locus.,” Int J Mol Med 16(2):355-358 (2005), which is herebyincorporated by reference in its entirety). See also Zhang et al.,“Receptor Specificity of the Fibroblast Growth Factor Family,” J BiolChem 281(23):15694-15700; Yu et al., “Analysis of the BiochemicalMechanisms for the Endocrine Actions of Fibroblast Growth Factor-23,”Endocrinology 146(11):4647-4656, which are hereby incorporated byreference in their entirety.

The number of principal FGFRs is increased from four to seven due to amajor tissue-specific alternative splicing event in the second half ofthe immunoglobulin-like domain 3 of FGFR1 to FGFR3, which createsepithelial lineage-specific b and mesenchymal lineage-specific cisoforms (Mohammadi et al., “Structural Basis for Fibroblast GrowthFactor Receptor Activation,” Cytokine Growth Factor Rev 16:107-137(2005) and Ornitz et al., “Fibroblast Growth Factors,” Genome Biol2(3):reviews3005.1-reviews3005.12 (2001), which are hereby incorporatedby reference in their entirety). Generally, the receptor-bindingspecificity of FGFs is divided along this major alternative splicing ofreceptors whereby FGFRb-interacting FGFs are produced by epithelialcells (Ornitz et al., “Fibroblast Growth Factors,” Genome Biol2(3):reviews3005.1-reviews3005.12 (2001), which is hereby incorporatedby reference in its entirety). These reciprocal expression patterns ofFGFs and FGFRs result in the establishment of a paracrineepithelial-mesenchymal signaling which is essential for properorganogenesis and patterning during development as well as tissuehomeostasis in the adult organism.

Based on phylogeny and sequence identity, FGFs are grouped into sevensubfamilies (Ornitz et al., “Fibroblast Growth Factors,” Genome Biol2(3):reviews3005.1-reviews3005.12 (2001), which is hereby incorporatedby reference in its entirety). The FGF core homology domain(approximately 120 amino acids long) is flanked by N- and C-terminalsequences that are highly variable in both length and primary sequence,particularly among different FGF subfamilies. The core region of FGF19shares the highest sequence identity with FGF21 (38%) and FGF23 (36%),and therefore, these ligands are considered to form a subfamily.

The nucleic acid and amino acid sequences for homo sapiens (human) FGF23may be found using the following reference sequence ID number onGenBank, NM_(—)020638. The human FGF23 gene coding sequence (1-251) hasa nucleotide sequence of SEQ ID NO: 2 as follows:

cggcaaaaag gagggaatcc agtctaggat cctcacacca gctacttgca agggagaaggaaaaggccag taaggcctgg gccaggagag tcccgacagg agtgtcaggt ttcaatctcagcaccagcca ctcagagcag ggcacgatgt tgggggcccg cctcaggctc tgggtctgtgccttgtgcag cgtctgcagc atgagcgtcc tcagagccta tcccaatgcc tccccactgctcggctccag ctggggtggc ctgatccacc tgtacacagc cacagccagg aacagctaccacctgcagat ccacaagaat ggccatgtgg atggcgcacc ccatcagacc atctacagtgccctgatgat cagatcagag gatgctggct ttgtggtgat tacaggtgtg atgagcagaagatacctctg catggatttc agaggcaaca tttttggatc acactatttc gacccggagaactgcaggtt ccaacaccag acgctggaaa acgggtacga cgtctaccac tctcctcagtatcacttcct ggtcagtctg ggccgggcga agagagcctt cctgccaggc atgaacccacccccgtactc ccagttcctg tcccggagga acgagatccc cctaattcac ttcaacacccccataccacg gcggcacacc cggagcgccg aggacgactc ggagcgggac cccctgaacgtgctgaagcc ccgggcccgg atgaccccgg ccccggcctc ctgttcacag gagctcccgagcgccgagga caacagcccg atggccagtg acccattagg ggtggtcagg ggcggtcgagtgaacacgca cgctggggga acgggcccgg aaggctgccg ccccttcgcc aagttcatctagggtcgctg gaagggcacc ctctttaacc catccctcag caaacgcagc tcttcccaaggaccaggtcc cttgacgttc cgaggatggg aaaggtgaca ggggcatgta tggaatttgctgcttctctg gggtcccttc cacaggaggt cctgtgagaa ccaacctttg aggcccaagtcatggggttt caccgccttc ctcactccat atagaacacc tttcccaata ggaaaccccaacaggtaaac tagaaatttc cccttcatga aggtagagag aaggggtctc tcccaacatatttctcttcc ttgtgcctct cctctttatc acttttaagc ataaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaagcagtg ggttcctgag ctcaagactt tgaaggtgta gggaagaggaaatcggagat cccagaagct tctccactgc cctatgcatt tatgttagat gccccgatcccactggcatt tgagtgtgca aaccttgaca ttaacagctg aatggggcaa gttgatgaaaacactacttt caagccttcg ttcttccttg agcatctctg gggaagagct gtcaaaagactggtggtagg ctggtgaaaa cttgacagct agacttgatg cttgctgaaa tgaggcaggaatcataatag aaaactcagc ctccctacag ggtgagcacc ttctgtctcg ctgtctccctctgtgcagcc acagccagag ggcccagaat ggccccactc tgttcccaag cagttcatgatacagcctca ccttttggcc ccatctctgg tttttgaaaa tttggtctaa ggaataaatagcttttacac tggctcacga aaatctgccc tgctagaatt tgcttttcaa aatggaaataaattccaact ctcctaagag gcatttaatt aaggctctac ttccaggttg agtaggaatccattctgaac aaactacaaa aatgtgactg ggaagggggc tttgagagac tgggactgctctgggttagg ttttctgtgg actgaaaaat cgtgtccttt tctctaaatg aagtggcatcaaggactcag ggggaaagaa atcaggggac atgttataga agttatgaaa agacaaccacatggtcaggc tcttgtctgt ggtctctagg gctctgcagc agcagtggct cttcgattagttaaaactct cctaggctga cacatctggg tctcaatccc cttggaaatt cttggtgcattaaatgaagc cttaccccat tactgcggtt cttcctgtaa gggggctcca ttttcctccctctctttaaa tgaccaccta aaggacagta tattaacaag caaagtcgat tcaacaacagcttcttccca gtcacttttt tttttctcac tgccatcaca tactaacctt atactttgatctattctttt tggttatgag agaaatgttg ggcaactgtt tttacctgat ggttttaagctgaacttgaa ggactggttc ctattctgaa acagtaaaac tatgtataat agtatatagccatgcatggc aaatatttta atatttctgt tttcatttcc tgttggaaat attatcctgcataatagcta ttggaggctc ctcagtgaaa gatcccaaaa ggattttggt ggaaaactagttgtaatctc acaaactcaa cactaccatc aggggttttc tttatggcaa agccaaaatagctcctacaa tttcttatat ccctcgtcat gtggcagtat ttatttattt atttggaagtttgcctatcc ttctatattt atagatattt ataaaaatgt aacccctttt tcctttcttctgtttaaaat aaaaataaaa tttatctcag cttctgttag cttatcctct ttgtagtactacttaaaagc atgtcggaat ataagaataa aaaggattat gggaggggaa cattagggaaatccagagaa ggcaaaattg aaaaaaagat tttagaattt taaaattttc aaagatttcttccattcata aggagactca atgattttaa ttgatctaga cagaattatt taagttttatcaatattgga tttctggt

As described above, reference sequence ID number on Genbank NM_(—)020638shows the nucleotide sequence for human FGF23 (i.e. SEQ ID NO:2) encodesa protein with the amino acid sequence of SEQ ID NO: 3 as follows:

mlgarlrlwvcalcsvcsmsvlraypnaspllgsswgglihlytatarnsyhlqihknghvdgaphqtiysalmirsedagfvvitgvmsrrylcmdfrgnifgshyfdpencrfqhqtlengydvyhspqyhflvslgrakraflpgmnpppysqflsrrneiplihfntpiprrhtrsaeddserdplnvlkprarmtpapascsqelpsaednspmasdplgvvrggrvnthaggtgpegcrpfakfi

Furthermore, Luethy et al. have cloned the FGF23 gene to produce atransgenic mouse that expresses the gene, and analyzed the phenotype ofthe mouse (WO 01/61007 to Luethy et al., which is hereby incorporated byreference in its entirety). See also U.S. Patent Application PublicationNo. 20050106755 to Zahradnik et al., which is hereby incorporated byreference in its entirety).

The nucleic acid and amino acid sequences for the Mus musculus (mouse)FGF23 may be found at GenBank, NM_(—)022657. The mouse FGF23 gene codingsequence has a nucleotide sequence SEQ ID NO: 4 as follows:

gaatctagcc caggatcccc acctcagttc tcagcttctt cctaggaaga agagaaaggccagcaagggc ccagcctgtc tgggagtgtc agatttcaaa ctcagcatta gccactcagtgctgtgcaat gctagggacc tgccttagac tcctggtggg cgtgctctgc actgtctgcagcttgggcac tgctagagcc tatccggaca cttccccatt gcttggctcc aactggggaagcctgaccca cctgtacacg gctacagcca ggaccagcta tcacctacag atccatagggatggtcatgt agatggcacc ccccatcaga ccatctacag tgccctgatg attacatcagaggacgccgg ctctgtggtg ataacaggag ccatgactcg aaggttcctt tgtatggatctccacggcaa catttttgga tcgcttcact tcagcccaga gaattgcaag ttccgccagtggacgctgga gaatggctat gacgtctact tgtcgcagaa gcatcactac ctggtgagcctgggccgcgc caagcgcatc ttccagccgg gcaccaaccc gccgcccttc tcccagttcctggctcgcag gaacgaggtc ccgctgctgc atttctacac tgttcgccca cggcgccacacgcgcagcgc cgaggaccca ccggagcgcg acccactgaa cgtgctcaag ccgcggccccgcgccacgcc tgtgcctgta tcctgctctc gcgagctgcc gagcgcagag gaaggtggccccgcagccag cgatcctctg ggggtgctgc gcagaggccg tggagatgct cgcgggggcgcgggaggcgc ggataggtgt cgcccctttc ccaggttcgt ctaggtcccc aggccaggctgcgtccgcct ccatcctcca gtcggttcag cccacgtaga ggaaggacta gggtacctcgaggatgtctg cttctctccc ttccctatgg gcctgagagt cacctgcgag gttccagccaggcaccgcta ttcagaatta agagccaacg gtgggaggct ggagaggtgg cgcagacagttctcagcacc cacaaatacc tgtaattcta gctccagggg aatctgtact cacacacacacacatccaca cacacacaca cacacataca tgtaatttta aatgttaatc tgatttaaagaccccaacag gtaaactaga cacgaagctc tttttatttt attttactaa caggtaaaccagacacttgg cctttattag ccgggtctct tgcctagcat tttaatcgat cagttagcacgaggaaagag ttcacgcctt gaacacaggg aagaggccat ctctgcagct tctagttactattctgggat tcacgggtgt ttgagtttga gcaccttgac cttaatgtct tcactaggcaagtcgaagaa agacgcgcat ttcttctctt tgggaagagc tttggattgg cgggaggctgacaaggacac ctaaaccgaa cacatttcag agttcagcct ccctgaggaa tgattcgccaatgattctgt gataggacca gtcagtagct tttgaatttg ccctggctca gcaaagtctaccttgctagg gtgttttgca aaatgcaaac gctcgaactc tctctaaaga ggcatttttagtgaaagcct ccgctagcag gttgacttgt aatatattct aagcgaatgt gcccggggtgggggtggagg tggggtgggg gagaagggtc cttgagacct cggattgttc taggttagggtttctgtgaa gagg

As described above, reference sequence ID number on Genbank NM_(—)022657shows the nucleotide sequence for mouse FGF23 (i.e. SEQ ID NO: 4)encodes a protein with the amino acid sequence of SEQ ID NO: 5 asfollows:

mlgtclrllvgvlctvcslgtaraypdtspllgsnwgslthlytatartsyhlqihrdghydgtphqtiysalmitsedagsvvitgamtrrflomdlhgnifgslhfspenckfrqwtlengydvylsqkhhylvslgrakrifqpgtnpppfsqflarrnevpllhfytvrprrhtrsaedpperdpinylkprpratpvpvscsrelpsaeeggpaasdplgylrrgrgdarggaggadrcrpfprfv

Kurosu et al. and Urakawa et al. have identified Klotho as an obligateco-receptor of FGF23 (Kurosu et al., “Regulation of Fibroblast GrowthFactor-23 Signaling by Klotho,” J Biol Chem 281(10):6120-6123 (2006);Urakawa et al., “Klotho Converts Canonical FGF Receptor Into a SpecificReceptor for FGF23,” Nature 444:770-774 (2006), which are herebyincorporated by reference in their entirety).

The nucleic acid and amino acid sequences for the human Klotho (i.e. SEQID NO: 6) gene may be found at GenBank, NM_(—)004795. The human Klothogene coding sequence has a nucleotide sequence of SEQ ID NO: 6 asfollows:

cgcgcagcat gcccgccagc gccccgccgc gccgcccgcg gccgccgccg ccgtcgctgtcgctgctgct ggtgctgctg ggcctgggcg gccgccgcct gcgtgcggag ccgggcgacggcgcgcagac ctgggcccgt ttctcgcggc ctcctgcccc cgaggccgcg ggcctcttccagggcacctt ccccgacggc ttcctctggg ccgtgggcag cgccgcctac cagaccgagggcggctggca gcagcacggc aagggtgcgt ccatctggga tacgttcacc caccaccccctggcaccccc gggagactcc cggaacgcca gtctgccgtt gggcgccccg tcgccgctgcagcccgccac cggggacgta gccagcgaca gctacaacaa cgtcttccgc gacacggaggcgctgcgcga gctcggggtc actcactacc gcttctccat ctcgtgggcg cgagtgctccccaatggcag cgcgggcgtc cccaaccgcg aggggctgcg ctactaccgg cgcctgctggagcggctgcg ggagctgggc gtgcagcccg tggtcaccct gtaccactgg gacctgccccagcgcctgca ggacgcctac ggcggctggg ccaaccgcgc cctggccgac cacttcagggattacgcgga gctctgcttc cgccacttcg gcggtcaggt caagtactgg atcaccatcgacaaccccta cgtggtggcc tggcacggct acgccaccgg gcgcctggcc cccggcatccggggcagccc gcggctcggg tacctggtgg cgcacaacct cctcctggct catgccaaagtctggcatct ctacaatact tctttccgtc ccactcaggg aggtcaggtg tccattgccctaagctctca ctggatcaat cctcgaagaa tgaccgacca cagcatcaaa gaatgtcaaaaatctctgga ctttgtacta ggttggtttg ccaaacccgt atttattgat ggtgactatcccgagagcat gaagaataac ctttcatcta ttctgcctga ttttactgaa tctgagaaaaagttcatcaa aggaactgct gacttttttg ctctttgctt tggacccacc ttgagttttcaacttttgga ccctcacatg aagttccgcc aattggaatc tcccaacctg aggcaactgctttcctggat tgaccttgaa tttaaccatc ctcaaatatt tattgtggaa aatggctggtttgtctcagg gaccaccaag agagatgatg ccaaatatat gtattacctc aaaaagttcatcatggaaac cttaaaagcc atcaagctgg atggggtgga tgtcatcggg tataccgcatggtccctcat ggatggtttc gagtggcaca gaggttacag catcaggcgt ggactcttctatgttgactt tctaagccag gacaagatgt tgttgccaaa gtcttcagcc ttgttctaccaaaagctgat agagaaaaat ggcttccctc ctttacctga aaatcagccc ctagaagggacatttccctg tgactttgct tggggagttg ttgacaacta cattcaagta gataccactctgtctcagtt taccgacctg aatgtttacc tgtgggatgt ccaccacagt aaaaggcttattaaagtgga tggggttgtg accaagaaga ggaaatccta ctgtgttgac tttgctgccatccagcccca gatcgcttta ctccaggaaa tgcacgttac acattttcgc ttctccctggactgggccct gattctccct ctgggtaacc agtcccaggt gaaccacacc atcctgcagtactatcgctg catggccagc gagcttgtcc gtgtcaacat caccccagtg gtggccctgtggcagcctat ggccccgaac caaggactgc cgcgcctcct ggccaggcag ggcgcctgggagaaccccta cactgccctg gcctttgcag agtatgcccg actgtgcttt caagagctcggccatcacgt caagctttgg ataacgatga atgagccgta tacaaggaat atgacatacagtgctggcca caaccttctg aaggcccatg ccctggcttg gcatgtgtac aatgaaaagtttaggcatgc tcagaatggg aaaatatcca tagccttgca ggctgattgg atagaacctgcctgcccttt ctcccaaaag gacaaagagg tggctgagag agttttggaa tttgacattggctggctggc tgagcccatt ttcggctctg gagattatcc atgggtgatg agggactggctgaaccaaag aaacaatttt cttcttcctt atttcactga agatgaaaaa aagctaatccagggtacctt tgactttttg gctttaagcc attataccac catccttgta gactcagaaaaagaagatcc aataaaatac aatgattacc tagaagtgca agaaatgacc gacatcacgtggctcaactc ccccagtcag gtggcggtag tgccctgggg gttgcgcaaa gtgctgaactggctgaagtt caagtacgga gacctcccca tgtacataat atccaatgga atcgatgacgggctgcatgc tgaggacgac cagctgaggg tgtattatat gcagaattac ataaacgaagctctcaaagc ccacatactg gatggtatca atctttgcgg atactttgct tattcgtttaacgaccgcac agctccgagg tttggcctct atcgttatgc tgcagatcag tttgagcccaaggcatccat gaaacattac aggaaaatta ttgacagcaa tggtttcccg ggcccagaaactctggaaag attttgtcca gaagaattca ccgtgtgtac tgagtgcagt ttttttcacacccgaaagtc tttactggct ttcatagctt ttctattttt tgcttctatt atttctctctcccttatatt ttactactcg aagaaaggca gaagaagtta caaatagttc tgaacatttttctattcatt cattttgaaa taattatgca gacacatcag ctgttaacca tttgcacctctaagtgttgt gaaactgtaa atttcataca tttgacttct agaaaacatt tttgtggcttatgacagagg ttttgaaatg ggcataggtg atcgtaaaat attgaataat gcgaatagtgcctgaatttg ttctcttttt gggtgattaa aaaactgaca ggcactataa tttctgtaacacactaacaa aagcatgaaa aataggaacc acaccaatgc aacatttgtg cagaaatttgaatgacaaga ttaggaatat tttcttctgc acccacttct aaatttaatg tttttctggaagtagtaatt gcaagagttc gaatagaaag ttatgtacca agtaaccatt tctcagctgccataataatg cctagtggct tcccctctgt caaatctagt ttcctatgga aaagaagatggcagatacag gagagacgac agagggtcct aggctggaat gttcctttcg aaagcaatgcttctatcaaa tactagtatt aatttatgta tctggttaat gacatacttg gagagcaaattatggaaatg tgtattttat atgatttttg aggtcctgtc taaaccctgt gtccctgagggatctgtctc actggcatct tgttgagggc cttgcacata ggaaactttt gataagtatctgcggaaaaa caaacatgaa tcctgtgata ttgggctctt caggaagcat aaagcaattgtgaaatacag tataccgcag tggctctagg tggaggaaag gaggaaaaag tgcttattatgtgcaacatt atgattaatc tgattataca ccatttttga gcagatcttg gaatgaatgacatgaccttt ccctagagaa taaggatgaa ataatcactc attctatgaa cagtgacactactttctatt ctttagctgt actgtaattt ctttgagttg atagttttac aaattcttaataggttcaaa agcaatctgg tctgaataac actggatttg tttctgtgat ctctgaggtctattttatgt ttttgctgct acttctgtgg aagtagcttt gaactagttt tactttgaactttcacgctg aaacatgcta gtgatatcta gaaagggcta attaggtctc atcctttaatgccccttaaa taagtcttgc tgattttcag acagggaagt ctctctatta cactggagctgttttataga taagtcaata ttgtatcagg caagataaac caatgtcata acaggcattgccaacctcac tgacacaggg tcatagtgta taataatata ctgtactata taatatatcatctttagagg tatgattttt tcatgaaaga taagcttttg gtaatattca ttttaaagtggacttattaa aattggatgc tagagaatca agtttatttt atgtatatat ttttctgattataagagtaa tatatgttca ttgtaaaaat ttttaaaaca cagaaactat atgcaaagaaaaaataaaaa ttatctataa tctcagaacc cagaaatagc cactattaac atttcctacgtattttattt tacatagatc atattgtata tagttagtat ctttattaat ttttattatgaaactttcct ttgtcattat tagtcttcaa aagcatgatt tttaatagtt gttgagtattccaccacagg aatgtatcac aacttaaccg ttcccgtttg ttagactagt ttcttattaatgttgatgaa tgttgtttaa aaataatttt gttgctacat ttactttaat ttccttgactgtaaagagaa gtaattttgc tccttgataa agtattatat taataataaa tctgcctgcaactttttgcc ttctttcata atcataaaaa aa

As described above, reference sequence ID number on Genbank NM_(—)004795shows the nucleotide sequence for human Klotho (i.e. SEQ ID NO: 6)encodes a protein with the amino acid sequence of SEQ ID NO: 7 asfollows:

dsrnaslplgapsplqpatgdvasdsynnvfrdtealrelgvthyrfsiswarvlpngsagvpnreglryyrrllerlrelgvqpvvtlyhwdlpqrlqdayggwanraladhfrdyaelcfrhfggqvkywitidnpyvvawhgyatgrlapgirgsprlgylvahnlllahakvwhlyntsfrptqggqvsialsshwinprrmtdhsikecqksldfvlgwfakpvfidgdypesmknnlssilpdftesekkfikgtadffalcfgptlsfqlldphmkfrqlespnlrqllswidlefnhpqifivengwfvsgttkrddakymyylkkfimetlkaikldgvdvigytawslmdgfewhrgysirrglfyvdflsqdkmllpkssalfyqkliekngfpplpenqplegtfpcdfawgvvdnyiqvdttlsqftdlnvylwdvhhskrlikvdgvvtkkrksycvdfaaiqpqiallqemhvthfrfsldwalilplgnqsqvnhtilqyyrcmaselvrvnitpvvalwqpmapnqglprllarqgawenpytalafaeyarlcfgelghhvklwitmnepytrnmtysaghnllkahalawhvynekfrhaqngkisialqadwiepacpfsqkdkevaervlefdigwlaepifgsgdypwvmrdwlnqrnnfllpyftedekkliqgtfdflalshyttilvdsekedpikyndylevqemtditwlnspsqvavvpwglrkvlnwlkfkygdlpmyiisngiddglhaeddqlrvyymqnyinealkahildginlcgyfaysfndrtaprfglyryaadqfepkasmkhyrkiidsngfpgpetlerfcpeeftvctecsffhtrksllafiaflffasiislslify yskkgrrsyk

The Klotho gene encodes a 130-kDa single-pass transmembrane protein witha short cytoplasmic domain (10 amino acids) and is expressedpredominantly in the kidney (Matsumara et al., “Identification of thehuman klotho gene and its two transcripts encoding membrane and secretedklotho protein,” Biochem Biophys Res Commun 242(3):626-630 (1998), whichis hereby incorporated by reference in its entirety). In addition to themembrane-bound isoform of Klotho, alternative splicing and proteolyticcleavage give rise to two soluble isoforms of Klotho found in thecirculation (Imura et al., “Secreted Klotho protein in sera and CSF:implication for post-translational cleavage in release of Klotho proteinfrom cell membrane,” FEBS Lett 565(1-3):143-147 (2004); Kurosu et al.,“Suppression of aging in mice by the hormone Klotho,” Science309(5742):1829-1833 (2005); Matsumura et al., “Identification of thehuman klotho gene and its two transcripts encoding membrane and secretedklotho protein,” Biochem Biophys Res Commun 242(3):626-630 (1998);Shiraki-Iida et al., “Structure of the mouse klotho gene and its twotranscripts encoding membrane and secreted protein,” FEBS Lett424(1-2):6-10 (1998), which are hereby incorporated by reference intheir entirety). Mice carrying a loss-of-function mutation in the Klothogene develop a syndrome resembling human aging, including shortened lifespan, skin atrophy, muscle atrophy, osteoporosis, arteriosclerosis, andpulmonary emphysema (Kuro-o et al., “Mutation of the Mouse Klotho GeneLeads to a Syndrome Resembling Ageing,” Nature 390:45-51 (1997), whichis hereby incorporated by reference in its entirety). Conversely,overexpression of the Klotho gene extends the life span and increasesresistance to oxidative stress in mice (Kurosu et al., “Suppression ofAging in Mice by the Hormone Klotho,” Science 309:1829-1833 (2005),which is hereby incorporated by reference in its entirety). Theseobservations suggest that the Klotho gene functions as an agingsuppressor gene.

The nucleic acid and amino acid sequences for the human FGFR1,transcript variant 1 gene may be found at GenBank, NM_(—)023110. TheFGFR1 has the nucleotide sequence of SEQ ID NO: 8 as follows:

agatgcaggg gcgcaaacgc caaaggagac caggctgtag gaagagaagg gcagagcgccggacagctcg gcccgctccc cgtcctttgg ggccgcggct ggggaactac aaggcccagcaggcagctgc agggggcgga ggcggaggag ggaccagcgc gggtgggagt gagagagcgagccctcgcgc cccgccggcg catagcgctc ggagcgctct tgcggccaca ggcgcggcgtcctcggcggc gggcggcagc tagcgggagc cgggacgccg gtgcagccgc agcgcgcggaggaacccggg tgtgccggga gctgggcggc cacgtccgga cgggaccgag acccctcgtagcgcattgcg gcgacctcgc cttccccggc cgcgagcgcg ccgctgcttg aaaagccgcggaacccaagg acttttctcc ggtccgagct cggggcgccc cgcagggcgc acggtacccgtgctgcagtc gggcacgccg cggcgccggg gcctccgcag ggcgatggag cccggtctgcaaggaaagtg aggcgccgcc gctgcgttct ggaggagggg ggcacaaggt ctggagaccccgggtggcgg acgggagccc tccccccgcc ccgcctccgg ggcaccagct ccggctccattgttcccgcc cgggctggag gcgccgagca ccgagcgccg ccgggagtcg agcgccggccgcggagctct tgcgaccccg ccaggacccg aacagagccc gggggcggcg ggccggagccggggacgcgg gcacacgccc gctcgcacaa gccacggcgg actctcccga ggcggaacctccacgccgag cgagggtcag tttgaaaagg aggatcgagc tcactgtgga gtatccatggagatgtggag ccttgtcacc aacctctaac tgcagaactg ggatgtggag ctggaagtgcctcctcttct gggctgtgct ggtcacagcc acactctgca ccgctaggcc gtccccgaccttgcctgaac aagcccagcc ctggggagcc cctgtggaag tggagtcctt cctggtccaccccggtgacc tgctgcagct tcgctgtcgg ctgcgggacg atgtgcagag catcaactggctgcgggacg gggtgcagct ggcggaaagc aaccgcaccc gcatcacagg ggaggaggtggaggtgcagg actccgtgcc cgcagactcc ggcctctatg cttgcgtaac cagcagcccctcgggcagtg acaccaccta cttctccgtc aatgtttcag atgctctccc ctcctcggaggatgatgatg atgatgatga ctcctcttca gaggagaaag aaacagataa caccaaaccaaaccgtatgc ccgtagctcc atattggaca tccccagaaa agatggaaaa gaaattgcatgcagtgccgg ctgccaagac agtgaagttc aaatgccctt ccagtgggac cccaaaccccacactgcgct ggttgaaaaa tggcaaagaa ttcaaacctg accacagaat tggaggctacaaggtccgtt atgccacctg gagcatcata atggactctg tggtgccctc tgacaagggcaactacacct gcattgtgga gaatgagtac ggcagcatca accacacata ccagctggatgtcgtggagc ggtcccctca ccggcccatc ctgcaagcag ggttgcccgc caacaaaacagtggccctgg gtagcaacgt ggagttcatg tgtaaggtgt acagtgaccc gcagccgcacatccagtggc taaagcacat cgaggtgaat gggagcaaga ttggcccaga caacctgccttatgtccaga tcttgaagac tgctggagtt aataccaccg acaaagagat ggaggtgcttcacttaagaa atgtctcctt tgaggacgca ggggagtata cgtgcttggc gggtaactctatcggactct cccatcactc tgcatggttg accgttctgg aagccctgga agagaggccggcagtgatga cctcgcccct gtacctggag atcatcatct attgcacagg ggccttcctcatctcctgca tggtggggtc ggtcatcgtc tacaagatga agagtggtac caagaagagtgacttccaca gccagatggc tgtgcacaag ctggccaaga gcatccctct gcgcagacaggtaacagtgt ctgctgactc cagtgcatcc atgaactctg gggttcttct ggttcggccatcacggctct cctccagtgg gactcccatg ctagcagggg tctctgagta tgagcttcccgaagaccctc gctgggagct gcctcgggac agactggtct taggcaaacc cctgggagagggctgctttg ggcaggtggt gttggcagag gctatcgggc tggacaagga caaacccaaccgtgtgacca aagtggctgt gaagatgttg aagtcggacg caacagagaa agacttgtcagacctgatct cagaaatgga gatgatgaag atgatcggga agcataagaa tatcatcaacctgctggggg cctgcacgca ggatggtccc ttgtatgtca tcgtggagta tgcctccaagggcaacctgc gggagtacct gcaggcccgg aggcccccag ggctggaata ctgctacaaccccagccaca acccagagga gcagctctcc tccaaggacc tggtgtcctg cgcctaccaggtggcccgag gcatggagta tctggcctcc aagaagtgca tacaccgaga cctggcagccaggaatgtcc tggtgacaga ggacaatgtg atgaagatag cagactttgg cctcgcacgggacattcacc acatcgacta ctataaaaag acaaccaacg gccgactgcc tgtgaagtggatggcacccg aggcattatt tgaccggatc tacacccacc agagtgatgt gtggtctttcggggtgctcc tgtgggagat cttcactctg ggcggctccc cataccccgg tgtgcctgtggaggaacttt tcaagctgct gaaggagggt caccgcatgg acaagcccag taactgcaccaacgagctgt acatgatgat gcgggactgc tggcatgcag tgccctcaca gagacccaccttcaagcagc tggtggaaga cctggaccgc atcgtggcct tgacctccaa ccaggagtacctggacctgt ccatgcccct ggaccagtac tcccccagct ttcccgacac ccggagctctacgtgctcct caggggagga ttccgtcttc tctcatgagc cgctgcccga ggagccctgcctgccccgac acccagccca gcttgccaat ggcggactca aacgccgctg actgccacccacacgccctc cccagactcc accgtcagct gtaaccctca cccacagccc ctgctgggcccaccacctgt ccgtccctgt cccctttcct gctggcagga gccggctgcc taccaggggccttcctgtgt ggcctgcctt caccccactc agctcacctc tccctccacc tcctctccacctgctggtga gaggtgcaaa gaggcagatc tttgctgcca gccacttcat cccctcccagatgttggacc aacacccctc cctgccacca ggcactgcct ggagggcagg gagtgggagccaatgaacag gcatgcaagt gagagcttcc tgagctttct cctgtcggtt tggtctgttttgccttcacc cataagcccc tcgcactctg gtggcaggtg ccttgtcctc agggctacagcagtagggag gtcagtgctt cgtgcctcga ttgaaggtga cctctgcccc agataggtggtgccagtggc ttattaattc cgatactagt ttgctttgct gaccaaatgc ctggtaccagaggatggtga ggcgaaggcc aggttggggg cagtgttgtg gccctggggc ccagccccaaactgggggct ctgtatatag ctatgaagaa aacacaaagt gtataaatct gagtatatatttacatgtct ttttaaaagg gtcgttacca gagatttacc catcgggtaa gatgctcctggtggctggga ggcatcagtt gctatatatt aaaaacaaaa aagaaaaaaa aggaaaatgtttttaaaaag gtcatatatt ttttgctact tttgctgttt tattttttta aattatgttctaaacctatt ttcagtttag gtccctcaat aaaaattgct gctgcttcat ttatctatgggctgtatgaa aagggtggga atgtccactg gaaagaaggg acacccacgg gccctggggctaggtctgtc ccgagggcac cgcatgctcc cggcgcaggt tccttgtaac ctcttcttcctaggtcctgc acccagacct cacgacgcac ctcctgcctc tccgctgctt ttggaaagtcagaaaaagaa gatgtctgct tcgagggcag gaaccccatc catgcagtag aggcgctgggcagagagtca aggcccagca gccatcgacc atggatggtt tcctccaagg aaaccggtggggttgggctg gggagggggc acctacctag gaatagccac ggggtagagc tacagtgattaagaggaaag caagggcgcg gttgctcacg cctgtaatcc cagcactttg ggacaccgaggtgggcagat cacttcaggt caggagtttg agaccagcct ggccaactta gtgaaaccccatctctacta aaaatgcaaa aattatccag gcatggtggc acacgcctgt aatcccagctccacaggagg ctgaggcaga atcccttgaa gctgggaggc ggaggttgca gtgagccgagattgcgccat tgcactccag cctgggcaac agagaaaaca aaaaggaaaa caaatgatgaaggtctgcag aaactgaaac ccagacatgt gtctgccccc tctatgtggg catggttttgccagtgcttc taagtgcagg agaacatgtc acctgaggct agttttgcat tcaggtccctggcttcgttt cttgttggta tgcctcccca gatcgtcctt cctgtatcca tgtgaccagactgtatttgt tgggactgtc gcagatcttg gcttcttaca gttcttcctg tccaaactccatcctgtccc tcaggaacgg ggggaaaatt ctccgaatgt ttttggtttt ttggctgcttggaatttact tctgccacct gctggtcatc actgtcctca ctaagtggat tctggctcccccgtacctca tggctcaaac taccactcct cagtcgctat attaaagctt atattttgctggattactgc taaatacaaa agaaagttca atatgttttc atttctgtag ggaaaatgggattgctgctt taaatttctg agctagggat tttttggcag ctgcagtgtt ggcgactattgtaaaattct ctttgtttct ctctgtaaat agcacctgct aacattacaa tttgtatttatgtttaaaga aggcatcatt tggtgaacag aactaggaaa tgaattttta gctcttaaaagcatttgctt tgagaccgca caggagtgtc tttccttgta aaacagtgat gataatttctgccttggccc taccttgaag caatgttgtg tgaagggatg aagaatctaa aagtcttcataagtccttgg gagaggtgct agaaaaatat aaggcactat cataattaca gtgatgtccttgctgttact actcaaatca cccacaaatt tccccaaaga ctgcgctagc tgtcaaataaaagacagtga aattgacctg aaaaaaaaaa aaaaaaa

As described above, reference sequence ID number on Genbank NM_(—)023110shows the nucleotide sequence for human FGFR1, transcript variant 1(i.e. SEQ ID NO: 8) encodes a protein with the amino acid sequence ofSEQ ID NO: 9 as follows:

mwswkcllfwavlvtatlctarpsptlpeqaqpwgapvevesflvhpgdllqlrcrlrddvqsinwlrdgvqlaesnrtritgeevevqdsvpadsglyacvtsspsgsdttyfsvnvsdalpssedddddddssseeketdntkpnrmpvapywtspekmekklhavpaaktvkfkcpssgtpnptlrwlkngkefkpdhriggykvryatwsiimdsvvpsdkgnytciveneygsinhtyqldvversphrpilqaglpanktvalgsnvefmckvysdpqphiqwlkhievngskigpdnlpyvqilktagvnttdkemevlhlrnvsfedageytclagnsiglshhsawltvlealeerpavmtsplyleiiiyctgafliscmvgsvivykmksgtkksdfhsqmavhklaksiplrrqvtvsadssasmnsgvllvrpsrlsssgtpmlagvseyelpedprwelprdrlvlgkplgegcfgqvvlaeaigldkdkpnrvtkvavkmlksdatekdlsdlisememmkmigkhkniinllgactqdgplyviveyaskgnlreylqarrppgleycynpshnpeeqlsskdlvscayqvargmeylaskkcihrdlaarnvlvtednvmkiadfglardihhidyykkttngrlpvkwmapealfdriythqsdvwsfgvllweiftlggspypgvpveelfkllkeghrmdkpsnotnelymmmrdcwhavpsqrptfkqlvedldrivaltsnqeyldlsmpldqyspsfpdtrsstcssgedsvfsheplpeepclprhpaglangglkrr

The protein encoded by this FGFR1, transcript variant 1 gene is a memberof the fibroblast growth factor receptor (FGFR) family, where amino acidsequences are highly conserved between members and throughout evolution.FGFR family members differ from one another in their ligand affinitiesand tissue distribution. A full-length representative protein consistsof an extracellular region, composed of three immunoglobulin-likedomains, a single hydrophobic membrane-spanning segment, and acytoplasmic tyrosine kinase domain. The extracellular portion of theprotein interacts with fibroblast growth factors, setting in motion acascade of downstream signals, ultimately influencing mitogenesis anddifferentiation. This particular family member binds both acidic andbasic fibroblast growth factors and is involved in limb induction.Mutations in this gene have been associated with Pfeiffer syndrome,Jackson-Weiss syndrome, Antley-Bixler syndrome, osteoglophonicdysplasia, and autosomal dominant Kallmann syndrome. See Itoh et al.,“The Complete Amino Acid Sequence of the Shorter Form of Human BasicFibroblast Growth Factor Receptor Deduced from its cDNA,” BiochemBiophys Res Commun 169(2): 680-685 (1990); Dode et al., “KallmannSyndrome: Fibroblast Growth Factor Signaling Insufficiency?” J Mol Med82(11):725-34 (2004); Coumoul et al., “Roles of FGF Receptors inMammalian Development and Congenital Diseases,” Birth Defects Res CEmbryo Today 69(4):286-304 (2003), which are hereby incorporated byreference in their entirety. Alternatively, spliced variants whichencode different protein isoforms have been described; however, not allvariants have been fully characterized.

The nucleic acid and amino acid sequences for FGFR1 variants 2-6 may befound using the following reference sequence ID numbers on GenBank:FGFR1, transcript variant 2 (NM_(—)015850), FGFR1, transcript variant 3(NM_(—)023105), FGFR1, transcript variant 4 (NM_(—)023106), FGFR1,transcript variant 5 (NM_(—)023107), FGFR1, transcript variant 6(NM_(—)023108), and FGFR1, transcript variant 9, (NM_(—)023111). Thesesequences are hereby incorporated by reference in their entirety.

Hypophosphatemia may be due to renal phosphate wasting (such as,autosomal dominant hypophosphatemic rickets (ADHR), X-linkedhypophosphatemia (XLH), autosomal recessive hypophosphatemic rickets(ARHR), fibrous dysplasia (FD), McCune-Albright syndrome complicated byfibrous dysplasia (MAS/FD), Jansen's metaphyseal chondrodysplasia(Jansen's Syndrome), autosomal dominant polycystic kidney disease(ADPKD), tumor-induced osteomalacia (TIO), and chronic metabolicacidosis), other inherited or acquired renal phosphate wastingdisorders, alcoholic and diabetic ketoacidosis, acute asthma, chronicobstructive pulmonary disease (COPD), drug treatment of COPD, sepsis,recovery from organ (in particular, kidney) transplantation, parenteraliron administration, salicylate intoxication, severe trauma, chronictreatment with sucralfate and/or antacids, mechanical ventilation,eating disorder (such as, anorexia nervosa and bulimia nervosa), or therefeeding syndrome.

For each method, Klotho can have a nucleotide sequence of SEQ ID NO:6and the FGF23 may have a nucleotide sequence of SEQ ID NO:2.

Administration of the inhibitor of FGF23-Klotho-FGF receptor complexformation may be carried out orally, parenterally, subcutaneously,intravenously, intramuscularly, intraperitoneally, by intranasalinstillation, by implantation, by intracavitary or intravesicalinstillation, intraocularly, intraarterially, intralesionally,transdermally, or by application to mucous membranes. The inhibitor maybe administered with a pharmaceutically-acceptable carrier.

For the purpose of the present invention the following terms are definedbelow.

The term “hypophosphatemia” refers to serum phosphate concentrationbelow the normal range of 2.2 to 4.9 mg/dl (Dwyer et al., “Severehypophosphatemia in postoperative patients,” Nutr Clin Pract7(6):279-283 (1992); Alon et al., “Calcimimetics as an adjuvanttreatment for familial hypophosphatemic rickets,” Clin J Am Soc Nephrol3(3):658-664 (2008), which are hereby incorporated by reference in theirentirety).

The term “renal phosphate wasting” refers to an inherited or acquiredcondition in which renal tubular reabsorption of phosphate is impaired.

The term “disease” or “disorder” is used interchangeably herein, andrefers to any alteration in state of the body or of some of the organs,interrupting or disturbing the performance of the functions and/orcausing symptoms such as discomfort, dysfunction, distress, or evendeath to the person afflicted or those in contact with a person. Adisease or disorder can also relate to a distemper, ailing, ailment,malady, disorder, sickness, illness, complaint, inderdisposion, oraffectation.

The terms “treat”, “treating”, “treatment” and the like are usedinterchangeably herein and mean obtaining a desired pharmacologicaland/or physiological effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of partially or completely curing a diseaseand/or adverse effect attributed the disease. “Treating” as used hereincovers treating a disease in a vertebrate and particularly a mammal andmost particularly a human, and includes: (a) preventing the disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it; (b) inhibiting the disease, i.e.arresting its development; or (c) relieving the disease, i.e. causingregression of the disease.

A “subject” can be any mammal, particularly farm animals, mammalianpets, and humans.

The inhibitor used to treat hypophosphatemia may be the C-terminal tailpeptide of FGF23. The C-terminal tail peptide of FGF23 has an amino acidsequence of SEQ ID NO:11 or SEQ ID NO:12.

The sequences of FGF23¹⁸⁰⁻²⁵¹, FGF23¹⁸⁰⁻²⁰⁵, and FGF23²⁸⁻²⁵¹ are listedin Table 1.

TABLE 1 Schematic representation of the structure  of FGF23 fragmentsName of Peptide Amino Acid Sequence FGF23²⁸⁻²⁵¹asp llgsswggli hlytatarns yhlqihkngh (SEQ ID vdgaphqtiy salmirseda gfvvitgvms NO: 10)rrylcmdfrg nifgshyfdp encrfqhqtl engydvyhsp qyhflvslgr akraflpgmnpppysqflsr rneiplihfn tpiprrhtr saeddserdpl nvlkprarmt papascsqelpsaednspma sdplgvvrgg rvnthaggtg pegcrpfakfi FGF23¹⁸⁰⁻²⁵¹s aeddserdpl nvlkprarmt papascsqel (SEQ ID psaednspma sdplgvvrgg rvnthaggtg NO: 11) pegcrpfakf i FGF23¹⁸⁰⁻²⁰⁵s aeddserdpl nvlkprarmt papas (SEQ ID NO: 12)

The invention is particularly directed toward targeting FGF23-Klotho-FGFreceptor complex formation which makes it possible to treat patientswhich have experienced hypophosphatemia associated with elevated ornormal FGF23 levels or which would be expected to experiencehypophosphatemia associated with elevated or normal FGF23 levels andthus is particularly directed towards preventing, inhibiting, orrelieving the effects of hypophosphatemia. A subject is “treated”provided the subject experiences a therapeutically detectable andbeneficial effect, which may be measured based on a variety of differentcriteria generally understood by those skilled in the art to bedesirable with respect to the treatment of diseases related tohypophosphatemia.

The compounds of the present invention can be administered alone or withsuitable pharmaceutical carriers, and can be in solid or liquid formsuch as, tablets, capsules, powders, solutions, suspensions, oremulsions.

The active compounds of the present invention may be orallyadministered, for example, with an inert diluent, or with an assimilableedible carrier, or they may be enclosed in hard or soft shell capsules,or they may be compressed into tablets, or they may be incorporateddirectly with the food of the diet. For oral therapeutic administration,these active compounds may be incorporated with excipients and used inthe form of tablets, capsules, elixirs, suspensions, syrups, and thelike. Such compositions and preparations should contain at least 0.1% ofactive compound. The percentage of the compound in these compositionsmay, of course, be varied and may conveniently be between about 2% toabout 60% of the weight of the unit. The amount of active compound insuch therapeutically useful compositions is such that a suitable dosagewill be obtained. Preferred compositions according to the presentinvention are prepared so that an oral dosage unit contains betweenabout 1 and 250 mg of active compound.

The tablets, capsules, and the like may also contain a binder such asgum tragacanth, acacia, corn starch, or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch, alginic acid; a lubricant such as magnesium stearate; and asweetening agent such as sucrose, lactose, or saccharin. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier, such as a fatty oil.

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar, or both. A syrup may contain, in addition to activeingredient, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye, and flavoring such as cherry or orange flavor.

These active compounds may also be administered parenterally. Solutionsor suspensions of these active compounds can be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof in oils. Illustrative oils are those ofpetroleum, animal, vegetable, or synthetic origin, for example, peanutoil, soybean oil, or mineral oil. In general, water, saline, aqueousdextrose and related sugar solution, and glycols such as, propyleneglycol or polyethylene glycol, are preferred liquid carriers,particularly for injectable solutions. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

The compounds of the present invention may also be administered directlyto the airways in the form of an aerosol. For use as aerosols, thecompounds of the present invention in solution or suspension may bepackaged in a pressurized aerosol container together with suitablepropellants, for example, hydrocarbon propellants like propane, butane,or isobutane with conventional adjuvants. The materials of the presentinvention also may be administered in a non-pressurized form such as ina nebulizer or atomizer.

The compounds of the present invention may also be administered directlyto the airways in the form of a dry powder. For use as a dry powder, thecompounds of the present invention may be administered by use of aninhaler. Exemplary inhalers include metered dose inhalers and drypowdered inhalers. A metered dose inhaler or “MDI” is a pressureresistant canister or container filled with a product such as apharmaceutical composition dissolved in a liquefied propellant ormicronized particles suspended in a liquefied propellant. The correctdosage of the composition is delivered to the patient. A dry powderinhaler is a system operable with a source of pressurized air to producedry powder particles of a pharmaceutical composition that is compactedinto a very small volume. For inhalation, the system has a plurality ofchambers or blisters each containing a single dose of the pharmaceuticalcomposition and a select element for releasing a single dose.

Suitable powder compositions include, by way of illustration, powderedpreparations of the active ingredients thoroughly intermixed withlactose or other inert powders acceptable for intrabronchialadministration. The powder compositions can be administered via anaerosol dispenser or encased in a breakable capsule which may beinserted by the patient into a device that punctures the capsule andblows the powder out in a steady stream suitable for inhalation. Thecompositions can include propellants, surfactants and co-solvents andmay be filled into conventional aerosol containers that are closed by asuitable metering valve.

A second aspect of the present invention relates to a method ofscreening for compounds suitable for treatment of hypophosphatemiaassociated with elevated or normal FGF23 levels. This method involvesproviding: FGF23, binary FGFR-Klotho complex, and one or more candidatecompounds. The FGF23, the FGFR-Klotho complex, and the candidatecompounds are combined under conditions effective for the FGF23 and thebinary FGFR-Klotho complex to form a ternary complex if present bythemselves. The candidate compounds, which prevent formation of thecomplex, are identified as being potentially suitable in treatinghypophosphatemia associated with elevated or normal FGF23 levels.

For this method, a plurality of candidate compounds may be tested.

The candidate compound is contacted with an assay system according tothe selected assay system and candidate compound. For example, in an invitro cell culture system, the candidate compound may be added directlyto the cell culture medium, or the cells may be transfected with thecandidate compound, etc.

Surface plasmon resonance (SPR) spectroscopy is an in vitro method usedto determine physical interaction between two or more proteins. SPRspectroscopy is useful for confirming the existence of a protein:proteininteraction predicted by other research techniques (e.g.,co-immunoprecipitation, yeast two-hybrid and density gradientcentrifugation). The minimal requirement for SPR spectroscopy is theavailability of purified proteins, one of which will be coupled to thesurface of a biosensor chip.

Size-exclusion chromatography is another in vitro method used todetermine physical interaction between two or more proteins.Size-exclusion chromatography is useful for confirming the existence ofa protein:protein interaction predicted by other research techniques(e.g., co-immunoprecipitation, yeast two-hybrid and density gradientcentrifugation). The minimal requirement for size-exclusionchromatography is the availability of purified proteins.

A pull-down assay is yet another in vitro method used to determinephysical interaction between two or more proteins. Pull-down assays areuseful for confirming the existence of a protein:protein interactionpredicted by other research techniques (e.g., co-immunoprecipitation,yeast two-hybrid and density gradient centrifugation). The minimalrequirement for a pull-down assay is the availability of a purified andtagged protein which will be used to capture and ‘pull-down’ aprotein-binding partner.

A variety of interaction or binding assays can be used to determine thatan agent specifically binds the binary FGFR-Klotho complex, such as theSPR interaction analysis described below. One aspect of the presentinvention utilizes SPR analysis of FGF23 protein/peptide binding to thebinary FGFR-Klotho complex. The SPR analysis involved FGF23protein/peptide immobilization by amine coupling on flow channels of achip. Proteins were injected over the chip at a flow rate of 50 μlmin⁻¹, and at the end of each protein injection (180 s), HBS-EP buffer(10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v)polysorbate 20; 50 μl min⁻¹) was flowed over the chip to monitordissociation for 180 s. The chip surface was then regenerated byinjecting 50 μl of 2.0 M NaCl in 10 mM sodium acetate, pH 4.5. Tocontrol for nonspecific binding, FHF1B, which shares structuralsimilarity with FGFs but does not exhibit any FGFR binding, was coupledto the control flow channel of the chip. For each protein injection overa FGF23 protein/peptide chip, the nonspecific responses from the FHF1Bcontrol flow channel were subtracted from the responses recorded for theflow channel onto which FGF23 protein/peptide was immobilized. Toanalyze FGF23 binding to the binary FGFR1c-Klotho complex, FGF23²⁸⁻²⁵¹was coupled to a chip, and increasing concentrations of 1:1 complex ofthe ectodomains of FGFR1c and Klotho in HBS-EP buffer were passed overthe chip. To measure binding of the C-terminal tail of FGF23 to thebinary FGFR1c-Klotho complex, FGF23¹⁸⁰⁻²⁵¹ was immobilized on a chip,and increasing concentrations of 1:1 complex of the ectodomains ofFGFR1c and Klotho in HBS-EP buffer were passed over the chip. To examinewhether the C-terminal tail of FGF23 can compete with full-length FGF23for binding to the binary FGFR1c-Klotho complex, FGF23²⁸⁻²⁵¹ wasimmobilized on a chip. Increasing concentrations of FGF23¹⁸⁰⁻²⁵¹ weremixed with a fixed concentration of 1:1 complex of the ectodomains ofFGFR1c and Klotho in HBS-EP buffer, and the mixtures were passed overthe chip. As a control, competition of FGF23 in solution withimmobilized FGF23 for binding to the binary FGFR1c-Klotho complex wasstudied. Increasing concentrations of FGF23²⁸⁻²⁵¹ were mixed with afixed concentration of 1:1 complex of the ectodomains of FGFR1c andKlotho in HBS-EP buffer, and the mixtures were passed over the FGF23chip. Competition of the FGF23 C-terminal tail peptide with full-lengthFGF23 for binding to the binary FGFR1c-Klotho complex was also studiedusing the “reverse” SPR assay format, where FGF23¹⁸⁰⁻²⁵¹ was immobilizedon a chip and mixtures of a fixed concentration of 1:1 complex of theectodomains of FGFR1c and Klotho with increasing concentrations ofFGF23²⁸⁻²⁵¹ were passed over the chip. As a control, competition ofFGF23 C-terminal tail peptide in solution with immobilized FGF23C-terminal tail peptide for binding to the binary FGFR1c-Klotho complexwas analyzed. Increasing concentrations of FGF23¹⁸⁰⁻²⁵¹ were mixed witha fixed concentration of 1:1 complex of the ectodomains of FGFR1c andKlotho in HBS-EP buffer, and the mixtures were passed over theFGF23¹⁸⁰⁻²⁵¹ chip. To verify the specificity of the interaction betweenthe FGF23 C-terminal tail and the FGFR1c-Klotho complex, FGF23²⁸⁻²⁵¹ wasimmobilized on a chip. Increasing concentrations of FGF21¹⁶⁸⁻²⁰⁹ weremixed with a fixed concentration of 1:1 complex of the ectodomains ofFGFR1c and Klotho in HBS-EP buffer, and the mixtures were passed overthe chip. In addition, the ability of the FGF23 C-terminal tail peptideto interfere with binary complex formation between βKlotho and eitherFGF19 or FGF21 was tested, as was its ability to interfere with ternarycomplex formation between βKlotho, FGFR, and either FGF19 or FGF21.FGF19²³⁻²¹⁶ and FGF21²⁹⁻²⁰⁹ were immobilized on two flow channels of achip. FGF23¹⁸⁰⁻²⁵¹ and the ectodomain of βKlotho were mixed at a molarratio of 2:1, and the mixture was injected over the chip. Next,FGF23¹⁸⁰⁻²⁵¹ and the 1:1 complex of the ectodomains of FGFR1c andβKlotho were mixed at a molar ratio of 10:1, and the mixture was passedover the FGF19/FGF21 chip. To examine whether a C-terminal FGF23 peptidecomprising the minimal binding epitope for the binary FGFR-Klothocomplex can compete with full-length FGF23 for binding to FGFR1c-Klotho,increasing concentrations of FGF23¹⁸⁰⁻²⁰⁵ were mixed with a fixedconcentration of 1:1 complex of the ectodomains of FGFR1c and Klotho inHBS-EP buffer, and the mixtures were passed over a chip onto whichFGF23²⁸⁻²⁵¹ had been immobilized.

Size-exclusion chromatography may also be used to determine that anagent specifically binds the binary FGFR-Klotho complex. One aspect ofthe present invention utilizes size-exclusion chromatography. Thesize-exclusion chromatography experiments were performed on a HiLoad™16/60 Superdex™ 200 prep grade column. Because of poor solubility ofFGF23 proteins and FGFR1c ectodomain in low salt buffer, the experimentswere carried out with 25 mM HEPES-NaOH buffer, pH7.5, containing 1.0 MNaCl. Sample injection volume was 0.3 to 1.0 ml, and the flow rate was1.0 ml min⁻¹. Protein retention times were determined by absorbance at280 nm. The column was calibrated with ferritin (440 kDa),immunoglobulin G (150 kDa), albumin (69.3 kDa), ovalbumin (44.3 kDa),and carbonic anhydrase (28.8 kDa). The void volume was determined usingblue dextran 2,000. To examine binding of FGF23 proteins to the 1:1binary complex of the ectodomains of FGFR1c and Klotho, FGFR1c-Klothocomplex was mixed with a slight molar excess of either FGF23²⁸⁻²⁵¹ orFGF23²⁸⁻¹⁷⁹ or FGF23¹⁸⁰⁻²⁵¹, and the mixtures were applied to thesize-exclusion column. The retention time of the FGFR1c-Klotho complexalone served as a reference point. Proteins of column peak fractionswere resolved on 14% SDS-polyacrylamide gels, and then stained withCoomassie Brilliant Blue R-250.

A pull-down assay may also be used to confirm the existence of aprotein:protein interaction (i.e. FGF23¹⁸⁰⁻²⁵¹ binding to the binaryFGFR-Klotho complex). One aspect of the present invention utilizespull-down assays. These assays involved subconfluent cultures of aHEK293 cell line ectopically expressing the FLAG-taggedmembrane-spanning form of murine Klotho, which were harvested and lysed.Cell lysate was incubated with FGF23²⁸⁻²⁵¹, FGF23²⁸⁻²⁰⁰, FGF23²⁸⁻¹⁷⁹,FGF23¹⁸⁰⁻²⁵¹, or protein sample buffer, and binary complexes of Klothoand endogenous FGFR were isolated from cell lysate using anti-FLAG M2agarose beads. Bead-bound proteins were resolved together with controls(FGF23 protein) on 14% SDS-polyacrylamide gels, transferred tonitrocellulose membranes, and labeled using horseradishperoxidase-conjugated India-HisProbe.

Co-immunoprecipitation may also be used to determine that an agentspecifically binds the binary FGFR-Klotho complex. One aspect of thepresent invention utilizes co-immunoprecipitation studies. Subconfluentcultures of a HEK293 cell line ectopically expressing the FLAG-taggedmembrane-spanning form of murine Klotho were transfected with expressionvectors for V5-tagged FGFR1c, FGFR3c, or FGFR4. Two days later, thecells were lysed, and FGFR-Klotho complexes were isolated from celllysate using anti-V5 agarose beads. The beads were then incubated witheither FGF23¹⁸⁰⁻²⁵¹ or FGF23²⁸⁻²⁵¹ alone, or with mixtures ofFGF23²⁸⁻²⁵¹ with either increasing FGF23¹⁸⁰⁻²⁵¹ or increasingFGF23¹⁸⁰⁻²⁰⁵. Bead-bound proteins were resolved on SDS-polyacrylamidegels, transferred to nitrocellulose membranes, and labeled usingantibodies to Klotho, FGF23, and V5 epitope tag.

Serum FGF23 level may be evaluated in an individual withhypophosphatemia by immunoassay. This includes two kinds of enzymelinked immunoabsorbant assay (ELISA): a full-length assay that detectsonly full-length FGF23 with phosphate-lowering activity and a C-terminalassay that measures full-length as well as C-terminal fragment of FGF23.The FGF23 gene may be analyzed by direct sequencing of PCR products, andmutant FGF23 may be analyzed by Western blotting using two kinds ofmonoclonal antibodies that recognize N- and C-terminal portion of theprocessing site of FGF23 after expression in mammalian cells.

In addition to full-length peptides, the present invention provides forpeptides having the biological activity of FGF23, as defined herein. Oneskilled in the art would appreciate, based on the sequences disclosedherein, that overlapping fragments of FGF23 can be generated usingstandard recombinant technology, for example, that described in Sambrooket al. (Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, New York, 1989) and Ausubel et al. (Current Protocolsin Molecular Biology, Green & Wiley, New York, 1997), which are herebyincorporated by reference in their entirety. One skilled in the artwould appreciate, based on the disclosure presented herein, that thebiological activity of FGF23 fragments could be tested by injecting thematerial into mice and evaluating whether injected mice exhibitincreased renal phosphate excretion and hypophosphatemia. Induction ofphosphate excretion and hypophosphatemia would serve as an indicationthat the FGF23 fragment retained biological activity. In addition, invitro assays can be used to test FGF23 biological activity. For example,isolated renal tubules may be perfused with FGF23 fragments andevaluated for alterations in phosphate transport, relative to wild-typeFGF23. Similarly, cell culture models which possess the necessary FGF23signal transduction machinery (i.e. FGF receptor 1, Klotho, and type IIsodium-dependent phosphate transporter) may be transfected with FGF23fragments and subsequently tested for alterations in phosphatetransport, relative to wild-type FGF23.

In situ hybridization assays are used to measure the level of expressionfor normal cells and suspected cells from a tissue sample. Labelling ofthe nucleic acid sequence allows for the detection and measurement ofrelative expression levels. By comparing the level of expression betweennormal cells and suspected cells from a tissue sample, candidatecompounds suitable for treatment of hypophosphatemia associated withelevated or normal FGF23 may be identified by the reduced expressionlevel of the gene product.

An approach to detecting the presence of a given sequence or sequencesin a polynucleotide sample involves selective amplification of thesequence(s) by polymerase chain reaction. PCR is described in U.S. Pat.No. 4,683,202 to Mullis et al. and Saiki et al., “EnzymaticAmplification of Beta-globin Genomic Sequences and Restriction SiteAnalysis for Diagnosis of Sickle Cell Anemia,” Science 230:1350-1354(1985), which are hereby incorporated by reference in their entirety. Inthis method, primers complementary to opposite end portions of theselected sequence(s) are used to promote, in conjunction with thermalcycling, successive rounds of primer-initiated replication. Theamplified sequence(s) may be readily identified by a variety oftechniques. This approach is particularly useful for detecting candidatecompounds suitable for treatment of hypophosphatemia associated withelevated or normal FGF23.

The present invention also relates to a method of screening thespecificity of compounds which prevent formation of theFGF23-Klotho-FGFR complex. This method involves providing FGF19,providing binary FGFR-βKlotho complex, and providing one or morecandidate compounds. The FGF19, the binary FGFR-βKlotho complex, and thecandidate compounds are combined under conditions effective for theFGF19 and the binary FGFR-βKlotho complex to form a ternary complex ifpresent by themselves. Candidate compounds which do not interfere withformation of the complex are identified as being specific andpotentially suitable in treating hypophosphatemia associated withelevated or normal FGF23 levels.

This aspect of the present invention is carried out with many of theprocedures described with respect to the screening method of the secondaspect of the present invention as described above. FGF19 can bereplaced with FGF21. The FGF receptor may have the amino acid sequenceof SEQ ID NO:9. This aspect of the present invention can be carried outusing surface plasmon resonance spectroscopy.

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention but are by no means intended to limit its scope.

Materials and Methods for Examples 1-8

Purification of FGF19, FGF21, FGF23, FGFR, Klotho and βKlotho Proteinsand Purification/Synthesis of FGF21 and FGF23 Peptides

Human FGF19 (R23 to K216, referred to as FGF19²³⁻²¹⁶), human FGF21 (H29to S209, referred to as FGF21²⁹⁻²⁰⁹), human FGF23 (A28 to I251, referredto as FGF23²⁸⁻²⁵¹; FIG. 1A) and C-terminally truncated FGF23 proteins(A28 to T200, referred to as FGF23²⁸⁻²⁰⁰; A28 to R179, referred to asFGF23²⁸⁻¹⁷⁹; FIG. 1A) were expressed in E. coli, refolded in vitro, andpurified by published protocols (Ibrahimi et al., “Biochemical Analysisof Pathogenic Ligand-dependent FGFR2 Mutations Suggests DistinctPathophysiological Mechanisms for Craniofacial and Limb Abnormalities,”Hum Mol Genet 13(19):2313-2324 (2004), Plotnikov et al., “CrystalStructures of Two FGF-FGFR Complexes Reveal the Determinants ofLigand-receptor Specificity,” Cell 101(4):413-424 (2000), which arehereby incorporated by reference in their entirety). In order tominimize proteolysis of FGF23²⁸⁻²⁵¹ and FGF23²⁸⁻²⁰⁰, arginine residues176 and 179 of the proteolytic cleavage site ¹⁷⁶RXXR¹⁷⁹ (SEQ ID NO: 1)were replaced with glutamine as it occurs in ADHR (Anonymous, “AutosomalDominant Hypophosphataemic Rickets is Associated with Mutations inFGF23,” Nat Genet 26(3):345-348 (2000); White et al.,“Autosomal-dominant Hypophosphatemic Rickets (ADHR) Mutations StabilizeFGF-23,” Kidney Int 60(6):2079-2086 (2001), which are herebyincorporated by reference in their entirety). The bacterially expressedFGF23²⁸⁻²⁵¹ protein exhibited similar bioactivity as full-length FGF23produced using a mammalian expression system, as judged by similarability of the two protein preparations to induce tyrosinephosphorylation of FRS2α and downstream activation of MAP kinase cascadein a HEK293 cell line ectopically expressing the membrane-spanning formof murine Klotho (Kurosu et al., “Regulation of fibroblast growthfactor-23 signaling by klotho,” J Biol Chem 281(10):6120-6123 (2006),which is hereby incorporated by reference in its entirety). Humanfibroblast growth factor homologous factor 1B (FHF1B) was purified by apublished protocol (Olsen et al., “Fibroblast growth factor (FGF)homologous factors share structural but not functional homology withFGFs,” J Biol Chem 278(36):34226-34236 (2003), which is herebyincorporated by reference in its entirety). Purified human FGF2 (M1 toS155) was obtained from Upstate Biotechnology. The ligand-binding domainof human FGFR1c (D142 to R365) was expressed in E. coli and purified bypublished protocols (Anonymous, “Autosomal Dominant HypophosphataemicRickets is Associated with Mutations in FGF23,” Nat Genet 26(3):345-348(2000); White et al., “Autosomal-dominant Hypophosphatemic Rickets(ADHR) Mutations Stabilize FGF-23,” Kidney Int 60(6):2079-2086 (2001),which are hereby incorporated by reference in their entirety). Theectodomain of murine Klotho (A35 to K982) was purified from culturemedia of a HEK293 cell line ectopically expressing the Klotho ectodomainas a fusion protein with a C-terminal FLAG tag (Kurosu et al.,“Regulation of fibroblast growth factor-23 signaling by klotho,” J BiolChem 281(10):6120-6123 (2006); Kurosu et al., “Suppression of aging inmice by the hormone Klotho,” Science 309(5742):1829-1833 (2005), whichare hereby incorporated by reference in their entirety). Similarly, theectodomain of murine βKlotho (F53 to L995) was expressed in HEK293 cellsas a fusion protein with a C-terminal FLAG tag and purified using thesame protocol as for the Klotho ectodomain. Purified bovineβ-glucuronidase was obtained from Sigma-Aldrich.

The N-terminally hexahistidine-tagged, 72-amino acid C-terminal tail ofhuman FGF23 (S180 to I251, referred to as FGF23¹⁸⁰⁻²⁵¹; FIG. 1A) wasexpressed in E. coli, and purified by nickel affinity-, ion-exchange-and size-exclusion chromatographies. A shorter peptide of the FGF23C-terminal region (S180 to S205, referred to as FGF23¹⁸⁰⁻²⁰⁵; FIG. 1A)was synthesized by solid phase synthesis (GenScript Corporation). TheN-terminally hexahistidine-tagged, 42-amino acid long C-terminal tail ofFGF21 (P168 to S209, referred to as FGF21¹⁶⁸⁻²⁰⁹) was expressed in E.coli, and purified by nickel affinity- and ion-exchangechromatographies.

Analysis of FGF23-FGFR1c-Klotho Interactions by Surface PlasmonResonance Spectroscopy

Surface plasmon resonance (SPR) spectroscopy experiments were performedon a Biacore 2000 instrument (Biacore AB), and FGF23-FGFR1c-Klothointeractions were studied at 25° C. in HBS-EP buffer (10 mM HEPES-NaOH,pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) polysorbate 20). Proteinswere immobilized by amine coupling on flow channels of research gradeCM5 chips (Biacore AB). Proteins were injected over a CM5 chip at a flowrate of 50 μl min⁻¹, and at the end of each protein injection (180 s),HBS-EP buffer (50 μl min⁻¹) was flowed over the chip to monitordissociation for 180 s. The chip surface was then regenerated byinjecting 50 μl of 2.0 M NaCl in 10 mM sodium acetate, pH 4.5. Tocontrol for nonspecific binding in experiments where Klotho ectodomainwas immobilized on the chip, β-glucuronidase was coupled to the controlflow channel of the chip (˜26-32 fmole/mm²). Like Klotho,β-glucuronidase is a member of family 1 glycosidases, and hencestructurally related to each of the two extracellular glycosidase-likedomains of Klotho. In experiments where FGF19, FGF21, FGF23 or theC-terminal tail of FGF23 were immobilized on the chip, FHF1B, whichshares structural similarity with FGFs but does not exhibit any FGFRbinding (Olsen et al., “Fibroblast growth factor (FGF) homologousfactors share structural but not functional homology with FGFs,” J BiolChem 278(36):34226-34236 (2003), which is hereby incorporated byreference in its entirety), was coupled to the control flow channel ofthe chip (˜14-71 fmole/mm²) The data were processed with BiaEvaluationsoftware (Biacore AB). For each protein injection over a Klotho chip,the nonspecific responses from the β-glucuronidase control flow channelwere subtracted from the responses recorded for the Klotho flow channel.Similarly, for each protein injection over a FGF chip, the nonspecificresponses from the FHF1B control flow channel were subtracted from theresponses recorded for the FGF flow channel. Each set of experiments wasrepeated at least three times, and for each experiment, at least twoprotein injections were repeated two to five times to monitor chipperformance and to verify reproducibility of the binding responses.

To analyze Klotho binding to FGFR1c, Klotho ectodomain was immobilizedon a chip (˜29-35 fmole/mm² of flow channel). Increasing concentrationsof FGFR1c ectodomain in HBS-EP buffer were injected over the chip.Maximal equilibrium responses were plotted against the concentrations ofFGFR1c ectodomain (FIG. 1B), and from the fitted saturation bindingcurve the equilibrium dissociation constant (K_(D)) was calculated. Thefitted binding curve was judged to be accurate based on the distributionof the residuals (even and near zero) and χ² (<10% of R_(max)).

To analyze FGF23 binding to both Klotho and FGFR1c alone, and to thebinary FGFR1c-Klotho complex, FGF23²⁸⁻²⁵¹ was coupled to a chip (˜16-53fmole/mm² of flow channel). To measure FGF23 binding to Klotho,increasing concentrations of Klotho ectodomain in HBS-EP buffer werepassed over the chip. To analyze FGF23 interaction with FGFR1c,increasing concentrations of FGFR1c ectodomain in HBS-EP buffer wereinjected over the chip. To measure FGF23 binding to the binaryFGFR1c-Klotho complex, increasing concentrations of 1:1 complex of theectodomains of FGFR1c and Klotho in HBS-EP buffer were passed over theFGF23 chip.

To analyze binding of the C-terminal tail of FGF23 to the binaryFGFR1c-Klotho complex, FGF23¹⁸⁰⁻²⁵¹ was immobilized on a chip (˜48fmole/mm² of flow channel), and increasing concentrations of 1:1 complexof the ectodomains of FGFR1c and Klotho in HBS-EP buffer were passedover the chip.

To examine whether the C-terminal tail of FGF23 can compete withfull-length FGF23 for binding to the binary FGFR1c-Klotho complex, twoassay formats were employed. In one assay, FGF23²⁸⁻²⁵¹ was immobilizedon a chip (˜16-53 fmole/mm² of flow channel). Increasing concentrationsof FGF23¹⁸⁰⁻²⁵¹ (0-400 nM) were mixed with a fixed concentration of 1:1complex of the ectodomains of FGFR1c and Klotho (10 nM, 15 nM and 20 nM,respectively) in HBS-EP buffer, and the mixtures were passed over thechip. As a control, competition of FGF23 in solution with immobilizedFGF23 for binding to the binary FGFR1c-Klotho complex was studied.Increasing concentrations of FGF23²⁸⁻²⁵¹ (0-50 nM) were mixed with afixed concentration of 1:1 complex of the ectodomains of FGFR1c andKlotho (15 nM and 20 nM, respectively) in HBS-EP buffer, and themixtures were passed over the FGF23 chip. In the other—reverse—assay,FGF23¹⁸⁰⁻²⁵¹ was immobilized on a chip (˜48.4 fmole/mm² of flowchannel). Increasing concentrations of FGF23²⁸⁻²⁵¹ (0-50 nM) were mixedwith a fixed concentration of 1:1 complex of the ectodomains of FGFR1cand Klotho (20 nM) in HBS-EP buffer, and the mixtures were passed overthe chip. As a control, competition of FGF23 C-terminal tail peptide insolution with immobilized FGF23 C-terminal tail peptide for binding tothe binary FGFR1c-Klotho complex was studied. Increasing concentrationsof FGF23¹⁸⁰⁻²⁵¹ (0-400 nM) were mixed with a fixed concentration of 1:1complex of the ectodomains of FGFR1c and Klotho (20 nM) in HBS-EPbuffer, and the mixtures were passed over the FGF23¹⁸⁰⁻²⁵¹ chip.

To examine whether a C-terminal FGF23 peptide comprising the minimalbinding epitope for the binary FGFR-Klotho complex can compete withfull-length FGF23 for binding to FGFR1c-Klotho, increasingconcentrations of FGF23¹⁸⁰⁻²⁰⁵ (0-800 nM) were mixed with a fixedconcentration of 1:1 complex of the ectodomains of FGFR1c and Klotho (15nM and 20 nM, respectively) in HBS-EP buffer, and the mixtures werepassed over a chip onto which FGF23²⁸⁻²⁵¹ had been immobilized (˜16fmole/mm² of flow channel).

To examine whether the C-terminal tail of FGF21 can compete withfull-length FGF23 for binding to binding to the binary FGFR1c-Klothocomplex, FGF23²⁸⁻²⁵¹ was immobilized on a chip (˜16 fmole/mm² of flowchannel). FGF21¹⁶⁸⁻²⁰⁹ was mixed with the 1:1 complex of the ectodomainsof FGFR1c and Klotho at molar ratios of 6:1 and 10:1, and the mixtureswere passed over the chip.

To examine whether the C-terminal tail peptide of FGF23 interferes withbinary complex formation between βKlotho and either FGF19 or FGF21,FGF19²³⁻²¹⁶ and FGF21²⁹⁻²⁰⁹ were immobilized on two flow channels of achip (˜29 fmole/mm² of flow channel). FGF23¹⁸⁰⁻²⁵¹ and the ectodomain ofβKlotho were mixed at a molar ratio of 2:1, and the mixture was injectedover the chip.

To examine whether the C-terminal tail peptide of FGF23 interferes withternary complex formation between βKlotho, FGFR, and either FGF19 orFGF21, FGF23¹⁸⁰⁻²⁵¹ and the 1:1 complex of the ectodomains of FGFR1c andβKlotho were mixed at a molar ratio of 10:1, and the mixture was passedover a chip onto which FGF19²³⁻²¹⁶ and FGF21²⁹⁻²⁰⁹ had been immobilized(˜29 fmole/mm² of flow channel).

Analysis of FGF23 Protein/Peptide Binding to FGFR1c-Klotho Complex bySize-Exclusion Chromatography

Size-exclusion chromatography experiments were performed on a HiLoad™16/60 Superdex™ 200 prep grade column (GE Healthcare) mounted on anÄKTApurifier (GE Healthcare). Because of poor solubility of FGF23proteins and FGFR1c ectodomain in low salt buffer, the experiments werecarried out with 25 mM HEPES-NaOH buffer, pH7.5, containing 1.0 M NaCl.Sample injection volume was 0.3 to 1.0 ml, and the flow rate was 1.0 mlmin⁻¹. Protein retention times were determined by absorbance at 280 nm.The column was calibrated with ferritin (440 kDa), immunoglobulin G (150kDa), albumin (69.3 kDa), ovalbumin (44.3 kDa), and carbonic anhydrase(28.8 kDa). The void volume was determined using blue dextran 2,000. Toexamine binding of FGF23 proteins to the 1:1 binary complex of theectodomains of FGFR1c and Klotho, 1.0 to 3.0 μmol of FGFR1c-Klothocomplex were mixed with a 3- to 5-fold molar excess of eitherFGF23²⁸⁻²⁵¹ or FGF23²⁸⁻¹⁷⁹ or FGF23¹⁸⁰⁻²⁵¹, and the mixtures wereapplied to the size-exclusion column. The retention time of theFGFR1c-Klotho complex alone served as a reference point. Proteins ofcolumn peak fractions were resolved on 14% SDS-polyacrylamide gels, andthen stained with Coomassie Brilliant Blue R-250.

Cell Culture-Pull-Down Assays of FGF23 Protein/Peptide Binding toFGFR-Klotho Complex

Subconfluent cultures of a HEK293 cell line ectopically expressing theFLAG-tagged membrane-spanning form of murine Klotho (HEK293-Klotho;Kurosu et al., “Regulation of Fibroblast Growth Factor-23 Signaling byKlotho,” J Biol Chem 281(10):6120-6123 (2006), which is herebyincorporated by reference in its entirety), were harvested and lysed(Goetz et al., “Molecular Insights into the Klotho-dependent, EndocrineMode of Action of Fibroblast Growth Factor 19 Subfamily Members,” MolCell Biol 27(9):3417-3428 (2007), which is hereby incorporated byreference in its entirety). Cell lysate was incubated with 2.7 nmoles ofFGF23²⁸⁻²⁵¹, FGF23²⁸⁻²⁰⁰, FGF23²⁸⁻¹⁷⁹, FGF23¹⁸⁰⁻²⁵¹, or protein samplebuffer, and binary complexes of Klotho and endogenous FGFR were isolatedfrom cell lysate using anti-FLAG M2 agarose beads (Sigma-Aldrich) (Goetzet al., “Molecular Insights into the Klotho-dependent, Endocrine Mode ofAction of Fibroblast Growth Factor 19 Subfamily Members,” Mol Cell Biol27(9):3417-3428 (2007), which is hereby incorporated by reference in itsentirety). Bead-bound proteins were resolved together with controls (130to 250 ng of each FGF23 protein) on 14% SDS-polyacrylamide gels,transferred to nitrocellulose membranes, and labeled using horseradishperoxidase-conjugated India-HisProbe (Pierce).

In parallel, subconfluent HEK293-Klotho cells (Kurosu et al.,“Regulation of Fibroblast Growth Factor-23 Signaling by Klotho,” J BiolChem 281(10):6120-6123 (2006), which is hereby incorporated by referencein its entirety) were transfected with expression vectors for V5-taggedFGFR1c, FGFR3c, or FGFR4 (Kurosu et al., “Regulation of FibroblastGrowth Factor-23 Signaling by Klotho,” J Biol Chem 281(10):6120-6123(2006), which is hereby incorporated by reference in its entirety) andbinding of FGF23 proteins/peptides to Klotho-FGFR complexes isolatedfrom cell lysate was analyzed. Two days later, the cells were lysed(Kurosu et al, “Suppression of Aging in Mice by the Hormone Klotho,”Science 309(5742):1829-1833 (2005), which is hereby incorporated byreference in its entirety), and FGFR-Klotho complexes were isolated fromcell lysate using anti-V5 agarose beads (Sigma-Aldrich) (Kurosu et al.,“Regulation of Fibroblast Growth Factor-23 Signaling by Klotho,” J BiolChem 281(10):6120-6123 (2006), which is hereby incorporated by referencein its entirety). The beads were then incubated with either FGF23¹⁸⁰⁻²⁵¹(1 nM) or FGF23²⁸⁻²⁵¹ (1 nM) alone, or with mixtures of FGF23²⁸⁻²⁵¹ (1nM) with either increasing FGF23¹⁸⁰⁻²⁵¹ (2 to 76 nM) or increasingFGF23¹⁸⁰⁻²⁰⁵ (0.1 to 10 μM). Bead-bound proteins were resolved onSDS-polyacrylamide gels, transferred to nitrocellulose membranes, andlabeled using antibodies to Klotho (KM2119, (Kato et al., “Establishmentof the Anti-Klotho Monoclonal Antibodies and Detection of Klotho Proteinin Kidneys,” Biochemical Biophysical Res Communications 267(2):597-602(2000), which is hereby incorporated by reference in its entirety)),FGF23 (R&D systems), and V5 epitope tag (Invitrogen).

Analysis of Phosphorylation of FRS2α and 44/42 MAP Kinase in EpithelialCell Lines

Subconfluent HEK293-Klotho cells (Kurosu et al., “Regulation ofFibroblast Growth Factor-23 Signaling by Klotho,” J Biol Chem281(10):6120-6123 (2006), which is hereby incorporated by reference inits entirety) were serum starved for 16 h and then stimulated for 10 minwith either FGF23²⁸⁻²⁵¹ (0.33 to 10 nM) or FGF23¹⁸⁰⁻²⁵¹ (0.76 to 76.3nM). In parallel experiments, cells were stimulated with FGF23²⁸⁻²⁵¹ (1nM) alone or with FGF23²⁸⁻²⁵¹ (1 nM) mixed with increasingconcentrations of either FGF23¹⁸⁰⁻²⁵¹ (0.76 to 76.3 nM) or FGF23¹⁸⁰⁻²⁰⁵(0.1 to 10 μM). Cell stimulation with FGF2 (2.9 nM) alone or FGF2 (2.9nM) mixed with increasing concentrations of FGF23¹⁸⁰⁻²⁵¹ (0.76 to 76.3nM) served as controls. Similarly, subconfluent cells of a CHO cell linestably expressing Klotho (Imura et al., “Secreted Klotho Protein in Seraand CSF: Implication for Post-translational Cleavage in Release ofKlotho Protein from Cell Membrane,” FEBS Lett 565(1-3):143-147 (2004),which is hereby incorporated by reference in its entirety) were treatedwith either FGF23²⁸⁻²⁵¹ (0.067 to 20 nM) or FGF23²⁸⁻²⁰⁰ (0.04 to 12 nM).

In a separate experiment, the biological activity of the bacteriallyexpressed FGF23²⁸⁻²⁵¹ protein was compared to that of FGF23²⁵⁻²⁵¹expressed in the mouse myeloma cell line NS0 (R&D Systems). SubconfluentHEK293-Klotho cells were serum starved, and then treated with either ofthe two FGF23 proteins.

After stimulation, the cells were lysed (Kurosu et al, “Suppression ofAging in Mice by the Hormone Klotho,” Science 309(5742):1829-1833(2005), which is hereby incorporated by reference in its entirety), andcellular proteins were resolved on SDS-polyacrylamide gels, transferredto nitrocellulose membranes, and the protein blots were probed withantibodies to phosphorylated FGF receptor substrate-2α (FRS2α),phosphorylated 44/42 MAP kinase and non-phosphorylated 44/42 MAP kinase,and Klotho. Except for the anti-Klotho antibody (Kato et al.,“Establishment of the Anti-Klotho Monoclonal Antibodies and Detection ofKlotho Protein in Kidneys,” Biochemical Biophysical Res Communications267(2):597-602 (2000), which is hereby incorporated by reference in itsentirety), all antibodies were from Cell Signaling Technology.

Measurement of Phosphate Uptake by Opossum Kidney Cells

The effects of FGF23 proteins/peptides on sodium-coupled phosphateuptake were studied in the opossum kidney cell line OKP (Miyauchi etal., “Stimulation of transient elevations in cytosolic Ca2+ is relatedto inhibition of Pi transport in OK cells,” Am J Physiol 259(3 Pt2):F485-493 (1990), which is hereby incorporated by reference in itsentirety). The cell line has many characteristics of renal proximaltubule epithelium, including sodium gradient-dependent phosphatetransport and sensitivity to parathyroid hormone (Miyauchi et al.,“Stimulation of transient elevations in cytosolic Ca2+ is related toinhibition of Pi transport in OK cells,” Am J Physiol 259(3 Pt2):F485-493 (1990), which is hereby incorporated by reference in itsentirety). OKP cells also express FGFR1-4 and Klotho (see next methodssection). OKP cells were grown in culture as described previously (Hu etal., “Dopamine Acutely Stimulates Na+/H+ Exchanger (NHE3) EndocytosisVia Clathrin-coated Vesicles: Dependence on Protein Kinase A-mediatedNHE3 Phosphorylation,” J Biol Chem 276(29):26906-26915 (2001), which ishereby incorporated by reference in its entirety). Cells grown in24-well plates were stimulated for 4 h with FGF23²⁸⁻²⁵¹ (0.5 to 1 nM),FGF23¹⁸⁰⁻²⁵¹ (500 nM), FGF23¹⁸⁰⁻²⁰⁵ (500 nM), or mixtures of FGF23²⁸⁻²⁵¹(1 nM) with either FGF23¹⁸⁰⁻²⁵¹ (1 to 500 nM) or FGF23¹⁸⁰⁻²⁰⁵ (1 to 500nM). The 1 nM concentration of FGF23²⁸⁻²⁵¹ was chosen for competitionexperiments with FGF23 C-terminal peptides because at thisconcentration, half-maximum inhibition of phosphate uptake is reached.After stimulation, the cells were rinsed with Na⁺-free solution followedby 5 min incubation with uptake solution containing 100 μM KH₂ ³²PO₄ (2mCi/ml, Perkin Elmer). The reaction was stopped by aspiration of uptakesolution and washing cells with ice-cold stop solution (10 mM HEPES pH7.4, 140 mM NaCl, 1 mM MgCl₂). Each transport reaction was performed intriplicates.

Analysis of FGFR and Klotho mRNA Expression in Opossum Kidney Cells

Total RNA was extracted from the OKP cell line (Miyauchi et al.,“Stimulation of transient elevations in cytosolic Ca2+ is related toinhibition of Pi transport in OK cells,” Am J Physiol 259(3 Pt2):F485-493 (1990), which is hereby incorporated by reference in itsentirety) using RNeasy kit (Qiagen). 5 μg of total RNA was used for cDNAsynthesis with random hexamer primers using SuperScript III First StrandSynthesis System (Invitrogen). FGFR1-4, Klotho, and β-actin transcriptswere detected by PCR using Platinum Taq DNA Polymerase (Invitrogen). ThePCR conditions were 94° C. for 1 min followed by 35 cycles of 95° C. for30 s, 54° C. for 30 s, and 72° C. for 60 s. The primers used were5′-TGATTTGCATTCTCCACCAA-3′ (SEQ ID NO: 13) and5′-CTTCTCCCCGCTTTTCTTCT-3′ (SEQ ID NO: 14) (FGFR1);5′-TATGGGCCAGATGGATTACC-3′ (SEQ ID NO: 15) and5′-GCACGTATACTCCCCAGCAT-3′ (SEQ ID NO: 16) (FGFR2);5′-ACCTGGTGTCCTGTGCCTAC-3′ (SEQ ID NO: 17) and5′-CATTCGATGGCCCTCTTTTA-3′ (SEQ ID NO: 18) (FGFR3);5′-CTGAAGCACATCGAGGTCAA-3′ (SEQ ID NO: 19) and5′-CCTGACTCCAGGGAGAACTG-3′ (SEQ ID NO: 20) (FGFR4);5′-AGCCCTCGAAAGATGACTGA-3′ (SEQ ID NO: 21) and5′-ACAAACCAGCCATTCTCCAC-3′ (SEQ ID NO: 22) (Klotho); and5′-GTGGGGGATGAGGCCCAGAG-3′ (SEQ ID NO: 23) and5′-AGCTGTGGTGGTGAAACTGT-3′ (SEQ ID NO: 24) (β-actin). PCR products wereresolved on 2% agarose gels containing ethidium bromide.

Measurement of Phosphate in Serum and Urine of Rodents

The phosphaturic activity of FGF23²⁸⁻²⁰⁰ was examined in ˜6-week oldC57BL/6 mice by a published protocol (Goetz et al., “Molecular Insightsinto the Klotho-dependent, Endocrine Mode of Action of Fibroblast GrowthFactor 19 Subfamily Members,” Mol Cell Biol 27(9):3417-3428 (2007),which is hereby incorporated by reference in its entirety). FGF23²⁸⁻²⁵¹,FGF23²⁸⁻²⁰⁰, or vehicle were injected IP into the animals. Each mousereceived two injections at 8 h intervals, of 5 μg of protein perinjection. Before the first injection and 8 h after the secondinjection, blood was drawn by cheek-pouch bleeding and spun at 3,000×gfor 10 min to obtain serum. Serum phosphate levels were determined usingPhosphorus Liqui-UV reagent (Stanbio Laboratory).

The anti-phosphaturic activity of FGF23 C-terminal peptides was examinedin normal Sprague-Dawley rats and in Hyp mice, a mouse model of humanX-linked hypophosphatemia (XLH) (Beck et al., “Pex/PEX TissueDistribution and Evidence for a Deletion in the 3′ Region of the PexGene in X-linked Hypophosphatemic Mice,” J Clin Invest 99(6):1200-1209(1997), Eicher et al., “Hypophosphatemia: Mouse Model for Human FamilialHypophosphatemic (Vitamin D-resistant) Rickets,” Proc Natl Acad Sci USA73(12):4667-4671 (1996), Strom et al., “Pex Gene Deletions in Gy and HypMice Provide Mouse Models for X-linked Hypophosphatemia,” Hum Mol Genet6(2):165-171 (1997), which are hereby incorporated by reference in theirentirety). The animals were fed a complete, fixed formula dietcontaining 0.94% phosphate. Anesthetized rats (220-250 g body weight)were administered IV either FGF23²⁸⁻²⁵¹ (0.1 μg kg body weight⁻¹) orFGF23¹⁸⁰⁻²⁵¹ (0.1 μg kg body weight⁻¹) or vehicle. Before and 3 h afterthe injection, blood was drawn from the carotid artery and urine wascollected through bladder catheterization. Plasma and urine chemistry ofanimals were analyzed using Vitros Chemistry Analyzer (Ortho-ClinicalDiagnosis). 10- to 15-week old Hyp mice were fasted for 8-12 h beforeadministering IP either FGF23¹⁸⁰⁻²⁵¹ (1 mg) or FGF23¹⁸⁰⁻²⁰⁵ (860 μg) orvehicle. Before and 2 h, 4 h, 8 h, and 24 h after the injection, urineand serum samples were collected. Phosphate concentrations in urine andserum were determined using Phosphorus Liqui-UV Test (StanbioLaboratory), and urine creatinine levels were measured using DetectX™Urinary Creatinine Detection Kit (LuminosAssays).

Analysis of NaP_(i)-2A and NaP_(i)-2C Protein Abundance in the ApicalBrush Border Membrane of Renal Proximal Tubule Epithelium

Immunoblot analysis of NaP_(i)-2A and NaP_(i)-2C protein abundance inrenal cortex tissue and isolated brush border membrane vesicles (BBMV),and NaP_(i)-2A immunostaining of renal tissue were performed asdescribed (Bacic et al., “Activation of Dopamine D1-like ReceptorsInduces Acute Internalization of the Renal Na⁺/phosphate CotransporterNaPi-IIa in Mouse Kidney and OK cells,” Am J Physiol Renal Physiol288(4):F740-747 (2005), Loffing et al., “Renal Na/H Exchanger NHE-3 andNa—PO₄ Cotransporter NaP_(i)-2 Protein Expression in GlucocorticoidExcess and Deficient States,” J Am Soc Nephrol 9(9):1560-1567 (1998),Moe et al., “Dietary NaCl Modulates Na(+)-H+ Antiporter Activity inRenal Cortical Apical Membrane Vesicles,” Am J Physiol 260(1 Pt2):F130-137 (1991), which are hereby incorporated by reference in theirentirety).

For immunoblot, rat kidney cortices were dissected and homogenized, andBBMV were isolated (Loffing et al., “Renal Na/H Exchanger NHE-3 andNa—PO₄ Cotransporter NaP_(i)-2 Protein Expression in GlucocorticoidExcess and Deficient States,” J Am Soc Nephrol 9(9):1560-1567 (1998),Moe et al., “Dietary NaCl Modulates Na(+)-H+ Antiporter Activity inRenal Cortical Apical Membrane Vesicles. Am J Physiol 260(1 Pt2):F130-137 (1991), which are hereby incorporated by reference in theirentirety). 30 μg of cortical/BBMV protein was solubilized in Laemmlisample buffer, fractionated by SDS-PAGE, transferred to PVDF membraneand labeled using polyclonal rabbit antibody for NaP_(i)-2A or -2C (kindgift from Drs. J. Biber and H. Murer, University of Zurich, Switzerland)(1:3,000 dilution) and monoclonal mouse antibody for β-actin (1:5,000dilution). For immunohistochemistry, rat kidneys were fixed in situ withperfusion of 2.5% paraformaldehyde via distal aorta of renal arteriesbefore nephrectomy. In some experiments, kidneys were harvested anddirectly frozen in Tissue TeK® OCT using liquid nitrogen, andcryosections (4 μm) were prepared and processed for immunofluorescentstaining (Bacic et al., “Activation of Dopamine D1-like ReceptorsInduces Acute Internalization of the Renal Na⁺/phosphate CotransporterNaPi-IIa in Mouse Kidney and OK cells,” Am J Physiol Renal Physiol288(4):F740-747 (2005), which is hereby incorporated by reference in itsentirety). Sections were incubated with polyclonal rabbit antibody forNaP_(i)-2A (1:300 dilution; kind gift from Dr. J. Biber) followed bysecondary antibodies conjugated to rhodamine (Molecular Probes). ForNaP_(i)-2A/β-actin double staining, the sections were then incubatedwith fluorescein isothiocyanate-phalloidin (1:50) (Molecular Probes) tostain β-actin filaments. Sections were visualized with a Zeiss LSM510microscope.

Statistical Analysis

Data are expressed as the mean±SE (n≧6 or more). Statistical analysiswas performed using Student's unpaired or paired t-test, or usinganalysis of variance (ANOVA) when applicable. A value of P≦0.05 wasconsidered as statistically significant.

Example 1 C-terminal Tail of FGF23 Mediates Binding of FGF23 to a denovo Site at the Composite FGFR1c-Klotho Interface

To understand how FGF23, FGFR and Klotho interact to form a ternarycomplex, the ternary complex was reconstituted in solution usingbioactive, full-length FGF23 (FGF23²⁸⁻²⁵¹; FIG. 1A), and the solubleectodomains of FGFR1c and Klotho. The binary complex of FGFR1cectodomain with Klotho ectodomain was formed by capturing the Klothoectodomain onto an FGFR1c affinity column from conditioned media of aHEK293 cell line ectopically expressing the Klotho ectodomain (Kurosu etal., “Regulation of Fibroblast Growth Factor-23 Signaling by Klotho,” JBiol Chem 281(10):6120-6123 (2006), which is hereby incorporated byreference in its entirety). The FGFR1c-Klotho complex was furtherpurified by size-exclusion chromatography to remove excess FGFR1c (FIG.1B). Next, the FGFR1c-Klotho complex was mixed with FGF23²⁸⁻²⁵¹, andternary complex formation was examined by size-exclusion chromatography.As shown in FIG. 1C, FGF23 co-eluted with the FGFR1c-Klotho complexdemonstrating that the ectodomains of FGFR1c and Klotho are sufficientto form a stable ternary complex with FGF23.

The size-exclusion data showing that Klotho and FGFR1c ectodomains forma stable binary complex (FIG. 1B) indicate that Klotho must harbor ahigh affinity binding site for FGFR1c. To further confirm this, surfaceplasmon resonance (SPR) spectroscopy was used to determine thedissociation constant of the FGFR1c-Klotho interaction. Klothoectodomain was immobilized on a biosensor chip, and increasingconcentrations FGFR1c ectodomain were passed over the chip. Consistentwith the results obtained using size-exclusion chromatography (FIG. 1B),Klotho bound FGFR1c with high affinity (K_(D)=72 nM; FIG. 1D). BecauseKlotho harbors a high affinity binding site for FGFR1c, it was reasonedthat Klotho might also possess a distinct high affinity binding site forFGF23, and promote FGF23-FGFR1c binding by engaging FGF23 and FGFR1csimultaneously. To test this, FGF23²⁸⁻²⁵¹ was coupled to a biosensorchip, and increasing concentrations of Klotho ectodomain were passedover the chip. As shown in FIG. 1F, Klotho bound poorly to FGF23²⁸⁻²⁵¹.These data demonstrate that the Klotho ectodomain contains a highaffinity binding site for FGFR1c but not for FGF23.

Next, binding of FGF23 to FGFR1c was measured by injecting increasingconcentrations of FGFR1c over the FGF23 chip. As shown in FIG. 1G,FGF23²⁸⁻²⁵¹ exhibited poor binding to FGFR1c. Thus, the SPR data showthat FGF23 exhibits poor binding affinity for both the Klotho ectodomainalone and the FGFR1c ectodomain alone. Together with the size-exclusionchromatography data showing that FGF23 binds stably to the purifiedbinary FGFR1c-Klotho complex, the data raised the question whether FGF23binds to a de novo site generated at the composite FGFR1c-Klothointerface. To test this, FGFR1c-Klotho complex was purified as describedabove, and increasing concentrations of the binary complex were passedover the FGF23 chip. As shown in FIG. 1E, FGF23²⁸⁻²⁵¹ bound to theFGFR1c-Klotho complex demonstrating that FGF23 interacts with a de novosite generated at the composite FGFR1c-Klotho interface.

It was then examined whether the C-terminal tail of FGF23 mediatesbinding of FGF23 to the FGFR1c-Klotho complex. To test this, theC-terminal tail peptide of FGF23 (FGF23¹⁸⁰⁻²⁵¹; FIG. 1A) was coupled toa biosensor chip and increasing concentrations of FGFR1c-Klotho complexwere passed over the chip. As shown in FIG. 2A, FGF23¹⁸⁰⁻²⁵¹ avidlybound to the binary complex. Size-exclusion chromatography andco-immunoprecipitation experiments yielded similar results supportingthe SPR data (FIGS. 2B, C, and D).

Example 2 C-Terminal Tail of FGF23 Competes with Full-Length FGF23 forBinding to the Binary FGFR-Klotho Complex

To fully nail down that the C-terminal tail of FGF23 mediates FGF23binding to the binary FGFR1c-Klotho complex, a fixed concentration ofFGFR1c-Klotho was mixed with increasing concentrations of FGF23¹⁸⁰⁻²⁵¹,and the mixtures were passed over the FGF23 chip. Mixtures ofFGF23²⁸⁻²⁵¹ with FGFR1c-Klotho were used as a control. As shown in FIGS.3A and D, FGF23¹⁸⁰⁻²⁵¹ competed, in a dose-dependent fashion, withFGF23²⁸⁻²⁵¹ for binding to the FGFR1c-Klotho complex. Half-maximuminhibition of FGFR1c-Klotho binding to FGF23²⁸⁻²⁵¹ was reached with a3.3-fold molar excess of FGF23¹⁸⁰⁻²⁵¹ over FGFR1c-Klotho complex (FIG.3D). As expected, less than an equimolar amount of FGF23²⁸⁻²⁵¹ relativeto FGFR1c-Klotho complex already yielded 50% inhibition of binding ofthe binary complex to immobilized FGF23²⁸⁻²⁵¹ (FIGS. 3C and D). Similarresults were obtained using the “reverse” SPR assay format, whereFGF23¹⁸⁰⁻²⁵¹ was immobilized on a chip and mixtures of a fixedconcentration of FGFR1c-Klotho complex with increasing concentrations ofFGF23²⁸⁻²⁵¹ were passed over the chip (FIG. 3E). Mixtures ofFGF23¹⁸⁰⁻²⁵¹ with FGFR1c-Klotho were used as a control (FIG. 3F). Toverify the specificity of the interaction between the FGF23 C-terminaltail and the FGFR1c-Klotho complex, the C-terminal tail peptide of FGF21and FGFR1c-Klotho were mixed at molar ratios of 6:1 and 10:1, and themixtures were injected over a FGF23 chip. As shown in FIG. 3G,FGF21¹⁶⁸⁻²⁰⁹ failed to inhibit binding of the FGFR1c-Klotho complex toimmobilized FGF23²⁸⁻²⁵¹. In addition, the ability of the FGF23C-terminal tail peptide to interfere with binary complex formationbetween βKlotho and either FGF19 or FGF21 was tested, as was its abilityto interfere with ternary complex formation between βKlotho, FGFR, andeither FGF19 or FGF21. FGF19²³⁻²¹⁶ and FGF21²⁹⁻²⁰⁹ were coupled to abiosensor chip, and a 2:1 mixture of FGF23¹⁸⁰⁻²⁵¹ and βKlotho ectodomainwas injected over the chip. As shown in FIGS. 4A and B, FGF23¹⁸⁰⁻²⁵¹failed to inhibit binding of βKlotho to immobilized FGF19 or FGF21.Likewise, a 10-fold molar excess of FGF23¹⁸⁰⁻²⁵¹ over FGFR1c-βKlotho didnot affect binding of the FGFR1c-βKlotho complex to immobilized FGF19 orFGF21 (FIGS. 4C and D). A co-immunoprecipitation based competition assayalso confirmed that the C-terminal tail peptide of FGF23 can inhibitbinding of FGF23 to its binary cognate FGFR-Klotho complex (FIG. 3H).Together, the data unambiguously demonstrate that the C-terminal tail ofFGF23 harbors the binding site for the binary FGFR-Klotho complex andhence is essential for formation of the ternary FGF23-FGFR-Klothocomplex. Importantly, the binding data unveil that proteolytic cleavageat the ¹⁷⁶RXXR¹⁷⁹ motif (SEQ ID NO:1) abrogates FGF23 activity byremoving the binding site for the binary FGFR-Klotho complex thatresides in the C-terminal tail of FGF23.

Example 3 Residues S180 to T200 of the C-Terminal Tail of FGF23 Comprisethe Minimal Binding Epitope for the FGFR-Klotho Complex

In follow-up studies, it was found that FGF23²⁸⁻²⁰⁰, which lacks thelast 51 C-terminal amino acids, still retains the ability toco-immunoprecipitate with the binary FGFR-Klotho complex (FIG. 2D). Thefinding suggested that FGF23²⁸⁻²⁰⁰ may have similar biological activityas the full-length protein. To test this, the ability of FGF23²⁸⁻²⁰⁰ andFGF23²⁸⁻²⁵¹ to induce tyrosine phosphorylation of FGF receptor substrate2α (FRS2α) and downstream activation of MAP kinase cascade inKlotho-expressing cultured cells, and to induce phosphaturia in mice,was examined. As shown in FIG. 5A, FGF23²⁸⁻²⁰⁰ induced phosphorylationof FRS2α and downstream activation of MAP kinase cascade at a dosecomparable to that of FGF23²⁸⁻²⁵¹. The truncated FGF23 was also nearlyas effective as the full-length ligand in reducing serum phosphateconcentration in healthy C57BL/6 mice (FIG. 5B). These data show thatdeletion of the last 51 amino acids from the FGF23 C-terminus has littleeffect on FGF23 biological activity, narrowing down the epitope on theFGF23 C-terminal tail for the composite FGFR-Klotho interface toresidues S180 and T200. Indeed, a FGF23 peptide comprising the minimalbinding epitope for FGFR-Klotho (FGF23¹⁸⁰⁻²⁰⁵; FIG. 1A) was able tocompete, in a dose-dependent fashion, with FGF23²⁸⁻²⁵¹ for binding tothe binary FGFR1c-Klotho complex (FIG. 3B). Half-maximum inhibition ofFGFR1c-Klotho binding to FGF23²⁸⁻²⁵¹ was reached with a 5.7-fold molarexcess of FGF23¹⁸⁰⁻²⁰⁵ over FGFR1c-Klotho complex (FIG. 3D). Similarly,in a co-immunoprecipitation based competition assay, the FGF23¹⁸⁰⁻²⁰⁵peptide was able to inhibit binding of FGF23 to the binary complexes ofits cognate FGFR and Klotho (FIG. 5C). The data also explain the findingby Garringer and colleagues showing that residues P189 to P203 arerequired for FGF23 signaling (Garringer et al., “Molecular genetic andbiochemical analyses of FGF23 mutations in familial tumoral calcinosis,”Am J Physiol Endocrinol Metab 295(4):E929-937 (2008), which is herebyincorporated by reference in its entirety).

Example 4 FGF23 C-Terminal Peptides Block FGF23 Signaling

Based on these data, it was postulated that FGF23¹⁸⁰⁻²⁵¹ andFGF23¹⁸⁰⁻²⁰⁵ should antagonize FGF23 signaling by competing withfull-length FGF23 for binding to the FGFR-Klotho complex. To test this,cells stably overexpressing Klotho were stimulated with FGF23²⁸⁻²⁵¹alone or FGF23²⁸⁻²⁵¹ mixed with increasing concentrations of eitherFGF23¹⁸⁰⁻²⁵¹ or FGF23¹⁸⁰⁻²⁰⁵. As shown in FIGS. 6A and B, both peptidesinhibited, in a dose-dependent fashion, FGF23-induced tyrosinephosphorylation of FRS2α and downstream activation of MAP kinasecascade.

To test the specificity of the FGF23 antagonists, the ability of theFGF23¹⁸⁰⁻²⁵¹ peptide to inhibit signaling of FGF2, a prototypicalparacrine-acting FGF, which does not require Klotho for signaling wasexamined. As shown in FIG. 6C, the FGF23 antagonist failed to inhibittyrosine phosphorylation of FRS2α and downstream activation of MAPkinase cascade induced by FGF2. These data show that FGF23 C-terminalpeptides specifically block FGF23 signaling.

Example 5 FGF23 C-Terminal Peptides Antagonize the Inhibitory Effect ofFGF23 on Sodium-Coupled Phosphate Uptake by Renal Proximal TubuleEpithelial Cells

In renal proximal tubule epithelium, FGF23 signaling leads to inhibitionof phosphate uptake. To establish further that FGF23 C-terminal peptidesblock FGF23 action, the effects of the peptides on sodium-coupledphosphate uptake in a proximal tubular cell model were studied. As shownin FIG. 7A, FGF23¹⁸⁰⁻²⁵¹ antagonized the inhibition of phosphate uptakeby FGF23²⁸⁻²⁵¹ in a dose-dependent fashion, with an IC₅₀ of about 21 nM.FGF23¹⁸⁰⁻²⁰⁵ exhibited a similar, albeit less potent antagonistic effect(FIG. 7B). As expected, neither of the two FGF23 C-terminal peptidesaltered phosphate uptake when applied alone (FIGS. 7A and B).

Example 6 FGF23 C-Terminal Peptides Antagonize Phosphaturic Activity ofFGF23 in Healthy Rats

These findings led to in vivo studies and an investigation of whetherthe FGF23 C-terminal peptides antagonize the phosphaturic effects ofendogenous FGF23. An IV injection of FGF23¹⁸⁰⁻²⁵¹ into healthySprague-Dawley rats led to renal phosphate retention, andhyperphosphatemia (FIG. 8), suggesting that FGF23 C-terminal peptidesantagonize the phosphaturic action of endogenous FGF23. As expected,injection of FGF23²⁸⁻²⁵¹ induced increases in excretion rate andfractional excretion of phosphate, and led to a significant decrease inplasma phosphate compared to vehicle-treated animals (FIG. 8).

FGF23 exerts its phosphaturic activity by inhibiting phosphate uptake byrenal proximal tubule epithelium. The effect has been attributed toreduced transport activity of NaP_(i)-2A and NaP_(i)-2C, reduced amountof NaP_(i)-2A and NaP_(i)-2C proteins in the apical brush bordermembrane, and at the more chronic level, repression of the NaP_(i)-2Aand NaP_(i)-2C genes (Baum et al., “Effect of Fibroblast GrowthFactor-23 on Phosphate Transport in Proximal Tubules,” Kidney Int68(3):1148-1153 (2005), Perwad et al., “Fibroblast Growth Factor 23Impairs Phosphorus and Vitamin D Metabolism In Vivo and Suppresses25-hydroxyvitamin D-1alpha-hydroxylase Expression In Vitro,” Am JPhysiol Renal Physiol 293(5):F1577-1583 (2007), Yamashita et al.,“Fibroblast Growth Factor (FGF)-23 Inhibits Renal Phosphate Reabsorptionby Activation of the Mitogen-activated Protein Kinase Pathway,” J BiolChem 277(31):28265-28270 (2002), Larsson et al., “Transgenic miceexpressing fibroblast growth factor 23 under the control of thealpha1(I) collagen promoter exhibit growth retardation, osteomalacia,and disturbed phosphate homeostasis,” Endocrinology 145(7):3087-3094(2004), Segawa et al., “Effect of hydrolysis-resistant FGF23-R179Q ondietary phosphate regulation of the renal type-II Na/Pi transporter,”Pflugers Arch 446(5):585-592 (2003), which are hereby incorporated byreference in their entirety). The abundance of NaP_(i)-2A protein inbrush border membrane vesicles isolated from the kidneys of rats wasexamined. An IV injection of FGF23¹⁸⁰⁻²⁵¹ into healthy rats led to anincrease in NaP_(i)-2A protein expression in the apical brush bordermembrane compared to vehicle treatment (FIGS. 9A and B). The peptideexhibited similar effects on the NaP_(i)-2C protein (FIG. 9C). Asexpected, injection of FGF23²⁸⁻²⁵¹ led to a decrease in NaP_(i)-2Aprotein expression (FIGS. 9A and B). These findings establish that FGF23C-terminal peptides counteract or cancel out FGF23's phosphaturic actionmediated through NaP_(i)-2A and NaP_(i)-2C.

Example 7 FGF23 C-Terminal Peptides Antagonize Phosphaturic Activity ofFGF23 in a Mouse Model of Renal Phosphate Wasting

To evaluate the therapeutic potential of FGF23¹⁸⁰⁻²⁵¹ for treating renalphosphate wasting, the peptide's efficacy in Hyp mice, a mouse model ofXLH (Anonymous., “A Gene (PEX) with Homologies to Endopeptidases isMutated in Patients with X-linked Hypophosphatemic Rickets. The HYPConsortium.,” Nat Genet 11(2):130-136 (1995); Beck et al., “Pex/PEXTissue Distribution and Evidence for a Deletion in the 3′ Region of thePex Gene in X-linked Hypophosphatemic Mice,” J Clin Invest99(6):1200-1209 (1997); Eicher et al., “Hypophosphatemia: Mouse Modelfor Human Familial Hypophosphatemic (Vitamin D-resistant) Rickets,” ProcNatl Acad Sci USA 73(12):4667-4671 (1996); Strom et al., “Pex GeneDeletions in Gy and Hyp Mice Provide Mouse Models for X-linkedHypophosphatemia,” Hum Mol Genet 6(2):165-171 (1997), which are herebyincorporated by reference in their entirety) was analyzed. XLH is aninherited phosphate wasting disorder associated with high FGF23, whichis thought to be due to reduced clearance of FGF23 from the circulation.Excess FGF23 causes increased phosphate excretion resulting inhypophosphatemia. As shown in FIG. 10, an IP injection of FGF23¹⁸⁰⁻²⁵¹induced a decrease in renal phosphate excretion in Hyp mice compared tovehicle treatment. The effect persisted for at least four hours postinjection. Concomitantly, serum phosphate levels were elevated by theFGF23 antagonist treatment (FIG. 10). Likewise, an IP injection of theFGF23¹⁸⁰⁻²⁰⁵ peptide, which comprises the minimal binding epitope forthe composite FGFR-Klotho interface, caused an increase in serumphosphate in Hyp mice compared to vehicle-treated animals (FIG. 10).These results show that FGF23 C-terminal peptides are effective inattenuating renal phosphate wasting caused by excess FGF23.

In the present invention, it was demonstrated that the proteolyticcleavage at the RXXR (SEQ ID NO:1) motif down-regulates FGF23 activityby a dual mechanism: by removing FGF23's binding site for the binaryFGFR-Klotho complex, and by generating an endogenous inhibitor of FGF23.This regulatory mechanism was exploited to develop a FGF23 antagonistwith therapeutic potential for hypophosphatemia associated with elevatedor normal FGF23.

Patients with phosphate wasting disorders are generally treatedsymptomatically, with oral phosphate supplementation and1,25-dihydroxyvitamin D₃/calcitriol. As alluded to in the background,oral phosphate therapy can be poorly tolerated, and in certaincircumstances can induce hyperparathyroidism and poses risk ofexacerbation of hypophosphatemia. In patients with XLH, the persistentand even exaggerated renal phosphate wasting during therapy can causenephrocalcinosis and nephrolithiasis. For patients with renal phosphatewasting from tumor-induced osteomalacia, a causative treatment optionexists, which is resection of the tumor producing excess amounts ofphosphaturic hormone. These tumors are often difficult to locate,however, or the tumors are found in locations that are difficult toaccess, leaving most patients with tumor-induced osteomalacia alsocurrently with no options other than symptomatic therapy (van Boekel etal., “Tumor Producing Fibroblast Growth Factor 23 Localized byTwo-staged Venous Sampling,” Eur J Endocrinol 158(3):431-437 (2008); Jande Beur S M., “Tumor-induced Osteomalacia,” JAMA 294(10):1260-1267(2005), which are hereby incorporated by reference in their entirety).Since excess FGF23 is the pathogenic factor in phosphate wastingdisorders, blocking its action with FGF23 C-terminal peptides holdspromise of providing the first causative pharmacotherapy.

In a mouse model of phosphate wasting disorders, it has been shown thatFGF23 C-terminal peptides are effective in counteracting thephosphaturic action of FGF23. The present invention warrants furtherevaluation of the peptides' efficacy in nonhuman primates, andeventually, in humans. Neutralizing FGF23 activity with antibodyprovides an alternative approach for treating renal phosphate wasting.Indeed, Aono, Yamazaki and colleagues have explored this approach, anddeveloped antibodies against FGF23 that effectively neutralize FGF23activity in both healthy mice and Hyp mice (Yamazaki et al., “Anti-FGF23Neutralizing Antibodies Show the Physiological Role and StructuralFeatures of FGF23,” J Bone Miner Res 23(9):1509-1518 (2008), Aono etal., “Therapeutic Effects of Anti-FGF23 Antibodies in HypophosphatemicRickets/Osteomalacia,” J Bone Miner Res, published online May 5^(th),DOI 10.1359/jmbr.090509 (2009), which are hereby incorporated byreference in their entirety).

While it has been conclusively demonstrated that the phosphaturicactivity of FGF23 is Klotho-dependent (Nakatani et al., “Inactivation ofklotho function induces hyperphosphatemia even in presence of high serumfibroblast growth factor 23 levels in a genetically engineeredhypophosphatemic (Hyp) mouse model,” FASEB J 23(11):3702-3711 (2009),which is hereby incorporated by reference in its entirety), thepossibility that FGF23 may have some Klotho-independent functions hasnot yet been ruled out experimentally. In this regard, the presentinvention of an inhibitory peptide approach may offer a more targetedtherapy for hypophosphatemia than anti-FGF23 antibodies as FGF23C-terminal peptides specifically target the binary FGFR-Klotho complexand hence only neutralize Klotho-dependent function of FGF23. Incontrast, the antibody approach does not discriminate betweenKlotho-dependent and -independent functions of FGF23. The FGF23C-terminal peptides can also serve as an experimental tool to dissectKlotho-dependent and -independent functions of FGF23. The ability of theFGF23 C-terminal peptides to specifically recognize the binary receptorcomplex makes them a powerful tool to image tissues that express thecognate FGFR-Klotho complexes of FGF23.

Hypophosphatemia complicates a wide variety of conditions such as therefeeding syndrome, diabetic ketoacidosis, asthma exacerbations andchronic obstructive pulmonary disease, and recovery from organ(particularly, kidney) transplantation (Gaasbeek et al.,“Hypophosphatemia: An Update on its Etiology and Treatment,” Am J Med118(10):1094-1101 (2005); Miller et al., “Hypophosphatemia in theEmergency Department Therapeutics,” Am J Emerg Med 18(4):457-461 (2000);Marinella M A., “Refeeding Syndrome and Hypophosphatemia,” J IntensiveCare Med 20(3):155-159 (2005), which are hereby incorporated byreference in their entirety). Indeed, hypophosphatemia complicatingrecovery from kidney transplantation, and parenteral iron therapy hasbeen associated with increased plasma levels of FGF23 (Bhan et al.,“Post-transplant hypophosphatemia: Tertiary ‘HyperPhosphatoninism’?”Kidney Int 70(8):1486-1494 (2006), Evenepoel et al., “Tertiary‘Hyperphosphatoninism’ accentuates hypophosphatemia and suppressescalcitriol levels in renal transplant recipients,” Am J Transplant7(5):1193-1200 (2007), Kawarazaki et al., “Persistent high level offibroblast growth factor 23 as a cause of post-renal transplanthypophosphatemia,” Clin Exp Nephrol 11(3):255-257 (2007), Trombetti etal., “FGF-23 and post-transplant hypophosphatemia: evidence for a causallink,” abstract number Su168 presented at the 30^(th) Annual Meeting ofthe American Society for Bone and Mineral Research (2008), Schouten etal., “FGF23 elevation and hypophosphatemia after intravenous ironpolymaltose: a prospective study,” J Clin Endocrinol Metab94(7):2332-2337 (2009), Shouten et al., “Iron polymaltose-induced FGF23elevation complicated by hypophosphataemic osteomalacia,” Ann ClinBiochem 46(2):167-169 (2009), Shimizu et al., “Hypophosphatemia inducedby intravenous administration of saccharated ferric oxide: another formof FGF23-related hypophosphatemia,” Bone 45(4):814-816 (2009), which arehereby incorporated by reference in their entirety). Thus, the FGF23antagonist discovered in the present invention may be of therapeuticvalue for a much broader collection of patients than phosphate wastingdisorders alone. The ability of FGF23 C-terminal peptides to enhancerenal phosphate retention in normal rats ushers in the option of usingthese peptides therapeutically in hypophosphatemic conditions whereFGF23 is not the primary cause of hypophosphatemia, and notdown-regulated as a compensatory mechanism.

Another indication for therapy with FGF23 C-terminal peptides, whichwould target still more patients than disorders complicated byhypophosphatemia, is chronic kidney disease, a condition with a growingincidence, currently affecting nearly 26 million people in the UnitedStates alone. Plasma levels of FGF23 increase as kidney functiondeclines in patients with chronic kidney disease (CKD) (Larsson et al.,“Circulating Concentration of FGF-23 Increases as Renal FunctionDeclines in Patients with Chronic Kidney Disease, But Does Not Change inResponse to Variation in Phosphate Intake in Healthy Volunteers,” KidneyInt 64(6):2272-2279 (2003), which is hereby incorporated by reference inits entirety), likely as a compensatory response to enhanced phosphateretention, and top 1000-fold of normal levels in patients with end-stageCKD (Gutierrez et al., “Fibroblast Growth Factor 23 and Mortality AmongPatients Undergoing Hemodialysis,” N Engl J Med 359(6):584-592 (2008);Jean et al., “High Levels of Serum Fibroblast Growth Factor (FGF)-23 areAssociated with Increased Mortality in Long Haemodialysis Patients,”Nephrol Dial Transplant 24(9):2792-2796 (2009), which are herebyincorporated by reference in their entirety). The gradual increases inplasma FGF23 correlate with disease progression (Fliser et al.,“Fibroblast Growth Factor 23 (FGF23) Predicts Progression of ChronicKidney Disease: the Mild to Moderate Kidney Disease (MMKD) Study,” J AmSoc Nephrol 18(9):2600-2608 (2007); Westerberg et al., “Regulation ofFibroblast Growth Factor-23 in Chronic Kidney Disease,” Nephrol DialTransplant 22(11):3202-3207 (2007), which are hereby incorporated byreference in their entirety), including suppression of 1,25-vitamin Dproduction and development of secondary hyperparathyroidism (Nakanishiet al., “Serum Fibroblast Growth Factor-23 Levels Predict the FutureRefractory Hyperparathyroidism in Dialysis Patients,” Kidney Int67(3):1171-1178 (2005); Shigematsu et al., “Possible Involvement ofCirculating Fibroblast Growth Factor 23 in the Development of SecondaryHyperparathyroidism Associated with Renal Insufficiency,” Am J KidneyDis 44(2):250-256 (2004), which are hereby incorporated by reference intheir entirety). Moreover, increased circulating FGF23 has emerged as anindependent risk factor for cardiovascular disease and mortality in CKD(Gutierrez et al., “Fibroblast Growth Factor 23 and Mortality AmongPatients Undergoing Hemodialysis,” N Engl J Med 359(6):584-592 (2008);Jean et al., “High Levels of Serum Fibroblast Growth Factor (FGF)-23 areAssociated with Increased Mortality in Long Haemodialysis Patients,”Nephrol Dial Transplant 24(9):2792-2796 (2009); Gutierrez et al.,“Fibroblast Growth Factor 23 and Left Ventricular Hypertrophy in ChronicKidney Disease,” Circulation 119(19):2545-2552 (2009); Mirza et al.,“Circulating Fibroblast Growth Factor-23 is Associated with VascularDysfunction in the Community,” Atherosclerosis 205(2):385-390 (2009);Mirza et al., “Serum Intact FGF23 Associate with Left Ventricular Mass,Hypertrophy and Geometry in an Elderly Population,” Atherosclerosis207(2):546-551 (2009); Nasrallah et al., “Fibroblast Growth Factor-23(FGF-23) is Independently Correlated to Aortic Calcification inHaemodialysis Patients,” Nephrol Dial Transplant 25(8):2679-2685 (2010),which are hereby incorporated by reference in their entirety),suggesting that FGF23 is implicated in the pathogenesis of CKD and itsadverse outcomes. Blocking FGF23 action with FGF23 C-terminal peptidesmay prove effective in preventing or attenuating the occurrence ofdisease complications such as hyperparathyroidism and vascularcalcification. Thus, the FGF23 antagonist of the present invention maybe of therapeutic value for a much broader collection of patients thanhypophosphatemia due to renal phosphate wasting alone.

The identification of the FGF23 C-terminal tail as a FGF23 antagonistsuggests that proteolytic cleavage not only removes the binding site onFGF23 for the FGFR-Klotho complex, but also generates an endogenousFGF23 antagonist. A pathophysiological role of the latter mechanism isindicated by familial tumoral calcinosis (FTC), an autosomal recessivemetabolic disorder with clinical manifestations opposing those ofphosphate wasting disorders. Missense mutations in either theUDP-N-acetyl-α-D-galactosamine:polypeptideN-acetylglactosaminyltransferase 3 (GALNT3) gene (Garringer et al., “TwoNovel GALNT3 Mutations in Familial Tumoral Calcinosis,” Am J Med Genet A143A(20):2390-2396 (2007); Ichikawa et al., “Tumoral CalcinosisPresenting with Eyelid Calcifications Due to Novel Missense Mutations inthe Glycosyl Transferase Domain of the GALNT3 Gene,” J Clin EndocrinolMetab 91(11):4472-4475 (2006); Topaz et al., “Mutations in GALNT3,Encoding a Protein Involved in O-linked Glycosylation, Cause FamilialTumoral Calcinosis,” Nat Genet 36(6):579-581 (2004); Dumitrescu et al.,“A Case of Familial Tumoral Calcinosis/hyperostosis-hyperphosphatemiaSyndrome Due to a Compound Heterozygous Mutation in GALNT3 DemonstratingNew Phenotypic Features,” Osteoporos Int (2008), which are herebyincorporated by reference in their entirety), or the FGF23 gene (Arayaet al., “A Novel Mutation in Fibroblast Growth Factor 23 Gene as a Causeof Tumoral Calcinosis,” J Clin Endocrinol Metab 90(10):5523-5527 (2005);Chefetz et al., “A Novel Homozygous Missense Mutation in FGF23 CausesFamilial Tumoral Calcinosis Associated with Disseminated VisceralCalcification,” Hum Genet 118(2):261-266 (2005); Larsson et al., “ANovel Recessive Mutation in Fibroblast Growth Factor-23 Causes FamilialTumoral Calcinosis,” J Clin Endocrinol Metab 90(4):2424-2427 (2005);Benet-Pages et al., “An FGF23 Missense Mutation Causes Familial TumoralCalcinosis with Hyperphosphatemia,” Hum Mol Genet 14(3):385-390 (2005),which are hereby incorporated by reference in their entirety), have beenassociated with FTC. All FTC patients have abnormally high plasma levelsof the C-terminal proteolytic fragment of FGF23 (Garringer et al., “TwoNovel GALNT3 Mutations in Familial Tumoral Calcinosis,” Am J Med Genet A143A(20):2390-2396 (2007); Ichikawa et al., “Tumoral CalcinosisPresenting with Eyelid Calcifications Due to Novel Missense Mutations inthe Glycosyl Transferase Domain of the GALNT3 Gene,” J Clin EndocrinolMetab 91(11):4472-4475 (2006); Topaz et al., “Mutations in GALNT3,Encoding a Protein Involved in O-linked Glycosylation, Cause FamilialTumoral Calcinosis,” Nat Genet 36(6):579-581 (2004); Dumitrescu et al.,“A Case of Familial Tumoral Calcinosis/hyperostosis-hyperphosphatemiaSyndrome Due to a Compound Heterozygous Mutation in GALNT3 DemonstratingNew Phenotypic Features,” Osteoporos Int (2008); Araya et al., “A NovelMutation in Fibroblast Growth Factor 23 Gene as a Cause of TumoralCalcinosis,” J Clin Endocrinol Metab 90(10):5523-5527 (2005); Chefetz etal., “A Novel Homozygous Missense Mutation in FGF23 Causes FamilialTumoral Calcinosis Associated with Disseminated Visceral Calcification,”Hum Genet 118(2):261-266 (2005); Larsson et al., “A Novel RecessiveMutation in Fibroblast Growth Factor-23 Causes Familial TumoralCalcinosis,” J Clin Endocrinol Metab 90(4):2424-2427 (2005), which arehereby incorporated by reference in their entirety). The presentinvention suggests that excess C-terminal FGF23 fragment may aggravatehyperphosphatemia, and the resulting soft tissue calcification, byantagonizing the action of any residual, functional FGF23 ligand inthese patients.

There has been a conundrum surrounding the mechanism of action of FGF23in the kidney because Klotho is expressed in the distal convolutedtubule (Kato et al., “Establishment of the anti-Klotho monoclonalantibodies and detection of Klotho protein in kidneys,” Biochem BiophysRes Commun 267(2):597-602 (2000), Li et al., “Immunohistochemicallocalization of Klotho protein in brain, kidney, and reproductive organsof mice,” Cell Struct Funct 29(4):91-99 (2004), Tsujikawa et al.,“Klotho, a gene related to a syndrome resembling human premature aging,functions in a negative regulatory circuit of vitamin D endocrinesystem,” Mol Endocrinol 17(12):2393-2403 (2003), which are herebyincorporated by reference in their entirety), whereas FGF23 inhibitsphosphate reabsorption in the proximal tubule (Baum et al., “Effect offibroblast growth factor-23 on phosphate transport in proximal tubules,”Kidney Int 68(3):1148-1153 (2005), Perwad et al., “Fibroblast growthfactor 23 impairs phosphorus and vitamin D metabolism in vivo andsuppresses 25-hydroxyvitamin D-1alpha-hydroxylase expression in vitro,”Am J Physiol Renal Physiol 293(5):F1577-F1583 (2007), Larsson et al.,“Transgenic mice expressing fibroblast growth factor 23 under thecontrol of the alpha1(I) collagen promoter exhibit growth retardation,osteomalacia, and disturbed phosphate homeostasis,” Endocrinology145(7):3087-3094 (2004), which are hereby incorporated by reference intheir entirety). A recent study suggested that FGF23 signaling initiatesin the distal tubule and its effects are then transmitted to theproximal tubule through an unknown diffusible paracrine factor (Farrowet al., “Initial FGF23-mediated signaling occurs in the distalconvoluted tubule,” J Am Soc Nephrol 20(5):955-960 (2009), which ishereby incorporated by reference in its entirety). In addition to themembrane-bound isoform of Klotho, alternative splicing and proteolyticcleavage give rise to two soluble isoforms of Klotho found in thecirculation (Imura et al., “Secreted Klotho protein in sera and CSF:implication for post-translational cleavage in release of Klotho proteinfrom cell membrane,” FEBS Lett 565(1-3):143-147 (2004), Kurosu et al.,“Suppression of aging in mice by the hormone Klotho,” Science309(5742):1829-1833 (2005), Matsumura et al., “Identification of thehuman klotho gene and its two transcripts encoding membrane and secretedklotho protein,” Biochem Biophys Res Commun 242(3):626-630 (1998),Shiraki-Iida et al., “Structure of the mouse klotho gene and its twotranscripts encoding membrane and secreted protein,” FEBS Lett424(1-2):6-10 (1998), which are hereby incorporated by reference intheir entirety). Importantly, the recombinant Klotho ectodomain that wasused to reconstitute the ternary FGF23-FGFR-Klotho complex in vitrocorresponds to the complete ectodomain of Klotho that is shed into thecirculation by a proteolytic cleavage at the juncture between theextracellular domain and transmembrane domain (Imura et al., “SecretedKlotho protein in sera and CSF: implication for post-translationalcleavage in release of Klotho protein from cell membrane,” FEBS Lett565(1-3):143-147 (2004), Kurosu et al., “Suppression of aging in mice bythe hormone Klotho,” Science 309(5742):1829-1833 (2005), which arehereby incorporated by reference in their entirety). Thus, the presentinvention points to the possibility that it is the shed soluble isoformof Klotho that makes its way to the proximal tubule to promote formationof FGF23-FGFR-Klotho ternary complex, and inhibition of phosphatereabsorption.

Example 8 The Isolated C-Terminal Tail of FGF23 Inhibits Renal PhosphateExcretion as an FGF23 Antagonist by Displacing FGF23 from its Receptor

FGF23 is an important phosphaturic hormone. FGF23 fragments wereexamined for binding to the binary FGFR-Klotho complex, FGFR activation,sodium-dependent phosphate transport, and phosphate balance. Based onFGF23 peptides (aa 28-251, 28-179, 28-200, 180-251, and 180-200) bindingto the binary FGFR-Klotho complex, the binding region was localized toaa 180-200 which provides the structural platform to design agonists andantagonists. Using FRS2α and 44/42 MAP kinase phosphorylation asreadouts for FGFR activation, it was found that FGF23²⁸⁻²⁰⁰ was anagonist while FGF23¹⁸⁰⁻²⁵¹ had no activity alone but functioned as anantagonist. Its antagonistic action was mediated by competitivelydisplacing FGF23 from its binary cognate FGFR-Klotho complex, and themajor region of antagonism was further refined to aa 180-205. Next itwas examined if FGF23¹⁸⁰⁻²⁵¹ is a functional antagonist in vivo. An IVinjection of FGF23²⁸⁻²⁵¹ into normal rats induced hypophosphatemiawhereas FGF23¹⁸⁰⁻²⁵¹ induced hyperphosphatemia. Excretion rate andfractional excretion of phosphate were increased by FGF23²⁸⁻²⁵¹ butdecreased by FGF23¹⁸⁰⁻²⁵¹. FGF23²⁸⁻²⁵¹ diminished the sodium-dependentphosphate transporter proteins NaP_(i)-2A and NaP_(i)-2C in the apicalbrush border membrane whereas FGF23¹⁸⁰⁻²⁵¹ increased NaP_(i)-2A andNaP_(i)-2C protein expression. To ensure that these are direct effectson epithelia of the renal proximal tubule, phosphate uptake was studiedin proximal tubule-like cells. FGF23 C-terminal peptides did not alterphosphate uptake by themselves but they completely reversed theinhibitory effect of FGF23 on phosphate uptake (aa 180-251: half max 21nM; aa 180-205: half max between 100 nM and 500 nM). In conclusion, theisolated C-terminal tail of FGF23 is an antagonist of FGF23 and inducesrenal phosphate retention. This can provide the foundation for potentialtherapeutic interventions of hypophosphatemia where FGF23 is notdown-regulated as a compensatory mechanism.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

What is claimed:
 1. A method of screening for compounds suitable fortreatment of a hypophosphatemic condition, said method comprising:providing a receptor binding fragment of FGF23 consisting of the aminoacid sequence of SEQ ID NO:12 or amino acid residues 1 to 21 of SEQ IDNO:11; providing binary FGFR-Klotho complex; providing one or morecandidate compounds; combining the receptor binding fragment of FGF23,the binary FGFR-Klotho complex, and the candidate compounds underconditions effective for the receptor binding fragment of FGF23 and thebinary FGFR-Klotho complex to form a ternary complex if present bythemselves; and identifying the candidate compounds which preventformation of the ternary complex and bind the binary FGFR-Klotho complexas being suitable in treating hypophosphatemia associated with elevatedor normal FGF23.
 2. The method according to claim 1, wherein the Klothohas the amino acid sequence of SEQ ID NO:7.
 3. The method according toclaim 1, wherein a plurality of candidate compounds are tested.
 4. Themethod according to claim 1, wherein the FGF receptor has the amino acidsequence of SEQ ID NO:9.
 5. The method according to claim 1, wherein themethod is carried out using surface plasmon resonance spectroscopy. 6.The method according to claim 1, wherein the method is carried out usingsize-exclusion chromatography.
 7. The method according to claim 1,wherein the method is carried out using a pull-down assay.
 8. The methodaccording to claim 1, wherein the method is carried out usingco-immunoprecipitation.
 9. The method according to claim 1, wherein themethod is carried out in a cellular assay.
 10. The method according toclaim 1, wherein the receptor binding fragment of FGF23 consists of theamino acid sequence of SEQ ID NO:12.
 11. The method according to claim1, wherein the receptor binding fragment of FGF23 consists of amino acidresidues 1 to 21 of SEQ ID NO:11.
 12. The method according to claim 1,wherein the one or more candidate compounds comprise a polypeptide. 13.The method according to claim 1, wherein the one or more candidatecompounds comprise an FGF23 fragment.